# Patent

## Title
Compositions and methods for altering the morphology of plants

## Abstract

Compounds, methods for producing them and methods for varying the morphology of plants are disclosed. More particularly, a SUN gene can be used to alter the shape of fruit in a plant such as a tomato plant.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of PCT application No. PCT/US2008/076936 filed Sep. 18, 2008 which claims priority to U.S. Provisional Application No. 60/994,349, filed Sep. 19, 2007, the disclosure of which is incorporated herein by reference.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with Government support and the Government has rights in this invention under the National Science Foundation Grants DBI 0227541 and DBI 0400811.


TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION
This invention relates to the field of plant biology, and to compositions and methods for modifying the phenotype of a plant.
BACKGROUND OF THE INVENTION
Altering a trait in a plant has long been a desired goal. For example, elongated fruit is an important property in tomato from cultural and agronomical points of view. Tomatoes that are mechanically harvested and used for canning as well as the preparation of sauces typically feature elongated and blocky fruits. These shape characters are important to prevent the tomatoes to roll of conveyer belts during machine harvesting. Whole rectangular shaped tomatoes fit better in a can than when they are round in shape. Furthermore, the recent development of new varieties for fresh consumption resulted in an expansion of novel fruit shapes in this class of tomatoes. Perhaps most notable of these are the grape tomatoes which feature the size of a cherry tomato but the fruits are oval instead of round shaped. In addition to other improved qualities such as flavor and aroma, the distinct shape of the fruit makes it easy for consumers to separate the cherry and the grape tomatoes. The elongated fruit shape features undoubtedly led to the rapid increase in popularity of grape tomato in the last five years.
The molecular bases underlying fruit shape variation in plant species are largely unknown. Fruit crops display tremendous diversity in the morphology of the reproductive organ in comparison to their wild relatives. Wild relatives of tomato bear small and round fruit, while cultivated types bear fruit of increased size and many diverse shapes including flattened, rectangular and blocky, oxheart, bell pepper, long pepper and pear forms. This morphological variation is controlled by genetic loci that have major as well as minor effects1-5.
A prevalent morphological feature that distinguishes many cultivated tomatoes from undomesticated accessions is elongated fruit shape. Three major loci affect this feature: ovate, fs8.1, and sun, residing on chromosomes 2, 8, and 7, respectively. OVATE, which confers an elongated pear shape to fruit encodes a protein that negatively regulates plant growth6. The locus fs8.1 imparts an elongated blocky shape to fruit, while sun imparts an elongated and tapered shape to fruit1, 4, 8.
The locus sun comprises a major QTL and explains 58% of the phenotypic variation associated with elongated fruit shape in an F2 population derived from elongated-fruited S. lycopersicum variety ‘Sun1642’ and the small round-fruited wild relative S. pimpinellifolium, accession LA15898. Fine mapping indicates that sun resides in a dynamic region of the tomato genome where a large inversion comprising half of the short arm of chromosome 7, and small-scale insertions, deletions and tandem duplications distinguish the species in the tomato clade9. One insertion, estimated to be 30 kb, is particularly noteworthy because it is present in Sun1642 but not in LA1589, and is linked to fruit shape9.
Structural variations of genomes, such as duplications, deletions, inversions, and translocations, are prevalent in man and some of these variants underlie diseases10-12. The structural variants are named copy number variants if they comprise a region larger than 500 bp-1 kb but smaller than 3-5 Mb10, 11. Although the molecular mechanisms facilitating genome rearrangements resulting in copy number variants are often unknown, non-allelic homologous recombination is most commonly proposed.
In plants, the occurrence and extent of copy number variants and the role this type of structural variation plays in affecting phenotypic diversity within a species are largely unknown. The lack of information about structural variation within plant species is due to the lack of complete whole genome sequence information of accessions within the same species.
In addition to non-allelic homologous recombination, transposing elements can also create structural variations of genomes13, 14. Most notably, the transposition can lead to dramatic changes in phenotype when these elements land in the gene thereby inactivating its function. In fact, the ability of transposable elements to knock out host gene activity has been used extensively in functional analyses studies in many species.
An unusual group of transposable elements was discovered recently which were found to harbor segments of the host's genome. Of particular note are the Helitron and Pack-MULE DNA transposable elements found in maize, rice, and many other species15-20. These elements are unusual in that they ferry host gene and gene fragments around and have the potential to create novel proteins and protein functions through domain shuffling. Also, certain types of retroelements have the potential to create novel genes by read-through transcription into host genes followed by transposition of the element and its transduced segment elsewhere in the genome. Although this type of transposition is not often described in plants, the L1 retroelement is thought to be responsible for transducing up to 1% of the human genome21. Another transposon-like mechanism with the ability to generate novel functions is through the fortuitous reverse transcription of host mRNAs and the subsequent random insertion of these cDNA molecules into the genome. Although it is generally thought that most of these so-called retrogenes are non-functional, they have the potential to generate considerable phenotypic diversity in both plant and animals22 and provide one of the mechanisms for gene family expansion over evolutionary time23.
However, despite the potential of these latter types of transposable elements to underlie phenotypic variation via either the creation of novel genes, altering their expression through repositioning them in different chromosomal contexts, or by generating small interfering RNA that participate in silencing of host genes13, 14, 24, documented examples of a change in phenotype as a direct result of these types of transposition do not exist.
SUMMARY OF THE INVENTION
In one broad aspect, there is provided herein certain novel compounds, methods for producing them and methods for varying the morphology of plants. In a particular aspect, there is provided herein an unusual and complex retrotransposition-mediated event in which the duplication and repositioning of a gene at once resulted in a novel fruit shape phenotype found in tomato.
In a broad aspect, there is provided herein a transgenic plant that expresses at least one polynucleotide described herein, where at least a part of the transgenic plant has an altered trait as compared to a non-transgenic plant or wild-type plant.
In another broad aspect, the altered trait is one or more of: sensitivity to hormone levels, altered shape of at least part of the plant, altered plant size; altered leaf shape; altered vegetable shape, altered fruit shape; at least partially parthenocarpic fruit, increased SUN levels, and decreased SUN levels. Also, in certain embodiments, the altered trait is an overexpression of at least a portion of one of the isolated polynucleotides wherein the altered trait comprised a parthenocarpic fruit. Also, in certain embodiments, the altered trait is an expression of at least a portion of one of the isolated polynucleotides wherein the altered trait comprises an elongated fruit shape.
In another broad aspect, there is provided herein a method for producing a transgenic plant having an altered trait as compared to a non-transgenic or wild-type.
In another broad aspect, there is provided herein a method for making a plant having at least one fruit having a shape that is different from a naturally occurring fruit, comprising transforming a SUN inverted repeat construct into a near isogenic line of the fruit, and growing the plant.
In another broad aspect, there is provided herein food and food products comprising the fruit of the plants described herein. In another aspect, there is provided herein a near isogenic line (NIL) comprising a Sun1642 background. In another aspect, there is provided herein a near isogenic line (NIL) comprising a LA1589 background. In another aspect, there is provided herein a 17.2 kb pHX2 construct containing IQD12, SDL1-like, HYP1 and nucleotides encoding the first 415 amino acids of HYP2. In another aspect, there is provided herein a 14 kb pHX4 construct containing IQD12 and terminating 180 nucleotides upstream of the SDL1-like stop codon.
In another broad aspect, there is provided herein a method for altering at least one of a leaf and fruit shape of a plant, comprising introducing and expressing the polypeptide in the plant wherein the expressing the polypeptide alters the shape of the leaf and/or fruit, as compared to a plant that does not express at least one of the polypeptides described herein.
In another broad aspect, there is provided herein an isolated host cell transformed with a vector comprising at least one polypeptide described herein.
In another broad aspect, there is provided herein a method for increasing the regeneration ability of a plant, wherein the method comprises the step of expressing at least one polypeptide described herein in a cell of a plant.
In another broad aspect, there is provided herein an agent for altering at least one trait of a plant, wherein the agent comprises at least one polypeptide described herein, or a vector thereof as an active ingredient.
In another broad aspect, there is provided herein a method for determining the ability of a plant cell to produce a fruit with an altered shape, wherein the method comprises detecting the expression of at least one polypeptide described herein or a protein expressed thereby in the plant cell.
In another broad aspect, there is provided herein a method for determining the ability of a plant cell to produce a fruit with an altered shape, comprising detecting the expression of the polypeptide in the plant cell.
In another broad aspect, there is provided herein a method for improving the ability of a plant to produce a fruit with an altered shape, comprising regulating the activity of at least one protein produced by expression of at least one polypeptide described herein in the plant.
Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention.
In this regard, no attempt is made to show the various aspects of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
This application patent may contain at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.
FIGS. 1A-1C show the high resolution fine-mapping positions sun to a 30 kb region on chromosome 7:
FIG. 1A shows the genetic mapping and fine mapping placed sun to a 0.2 cM interval between two markers Lp81B9-F and Lp61O2-F. The vertical red (thick) bar defined the sun locus.
FIG. 1B shows the physical map of the sun locus using bacteriophage λ clones spanning the region. The upper portion displays the physical map of the S. lycopersicum Sun1642 genome and the lower portion displays the physical map of the S. pimpinellifolium LA1589 genome. The thin lines above and below the physical maps represent the phage clones. The diagonal dashed lines between Sun1642 and LA1589 physical maps indicate the colinearity between Sun1642 and LA1589 and also show where the 24.3 kb insertion occurs relative to the LA1589 physical map. The smallest region encompassing sun is indicated by the red bar and was defined by the recombination breakpoints flanking the locus. Thus, the sun locus maps to the short arm of tomato chromosome 7 between markers Lp81B9-F and Lp61O2-R and the recombination breakpoints delineate the locus (shown in red (thick line)) which is tightly associated with a 24.3 kb duplication.
FIG. 1C shows the structural organization the chromosome 7 sun locus (upper) and the chromosome 10 ancestral locus (lower). IQD12 (also referred to herein as SUN) is located 20 kb downstream of Rider in the transposed copy as opposed to 1 kb upstream of Rider at its ancestral position. The transposed 24.3 kb segment landed in the intron of DEFL1. Arrows show directionality of the predicted genes and pseudo-genes. Dark green arrows (HYP1, HYP2, SDL1-like) indicate ab initio predicted genes, purple arrows indicate the rearranged IQD12 gene, light green arrows indicate the retroelement Rider, and the yellow arrows (upper first box, second arrow, last arrow) indicate pseudo-genes. Red numbered boxes identify Rider's Long Terminal Repeats (LTR) and are numbered according to the order of transcription. The target site duplication (TSD) caused by Rider's original insertion into chromosome 10 is in green text (GACCT); the TSD resulting from the transposed duplication into chromosome 7 is in blue text (ATATT). PPT and PPT* indicate the position of the polypurine tract required for second strand synthesis of the retroelement.
FIGS. 2A-2C show that sun affects fruit shape post anthesis and in a dosage dependent manner. The effect of sun and candidate gene expression was determined using Near Isogenic Lines (NIL) differing for the presence and absence of the Sun1642 allele in the LA1589 background. The nearly Isogenic Lines (NILs) differing at sun display a large difference in fruit shape in young developing fruit 5 days after pollination. In those fruit, IQD12 is expressed at higher levels in oval shaped fruit due to increased expression at its new location on chromosome 7.
FIG. 2A shows that representative ovaries at 10 days pre anthesis, at anthesis, and at 5 days post anthesis from elongate-shaped, homozygous Sun1642 (ee) genotypes, and round-shaped homozygous LA1589 (pp) genotypes. Scale bar, 100 μm, 500 μm and 500 μm for images at pre anthesis, anthesis and post anthesis, respectively.
FIG. 2B shows that ovary shape index determined by calculating the ratio of organ length to organ width, were similar at 10 days pre anthesis (p=0.64, n=10 of ovaries per genotype). At anthesis, ovary shape index of the NILs was significantly different albeit small (p=0.04, n=5 plants of 8 ovaries per plant per genotype), whereas fruit shape index was highly significantly different at 5 days post anthesis (p<0.0001, n=5 plants comprising 6 to 8 fruits per plant per genotype).
FIG. 2C shows the Northern blot analyses of the five candidate genes at sun. Total RNA was isolated from pooled tissues from 5 plants of each genotype at the same developmental stages shown in FIG. 2A. Fragments of the candidate genes were sequentially hybridized to the Northern blot. The signal for eIF4α6 served as an RNA loading control. “pre”, tissue harvested 10 days pre anthesis; “ant”, tissue harvested at anthesis; “post”, tissue harvested 5 days post anthesis.
FIG. 2D shows that IQD12 and DEFL1 transcript levels are allele-specific, and dosage-dependent, and correlate with mature-stage fruit shape index. The fruit shape index of mature LA1589 fruit harboring 0, 1 or 2 copies of the Sun1642 allele is indicated above the graph bars. The average relative transcript levels of IQD12 and DEFL1 per genotype was determined by normalizing to eIF4α6 expression levels and was calculated for fruit harvested 5 days post pollination from 5 individual plants of each genotype (the Northern blot is shown in FIG. 9A). Error bars denote the standard deviation. Designations “ee” denote homozygous Sun1642; “pp” homozygous LA1589; “ep” heterozygous.
FIGS. 3A-3F show that tomato fruit shape is controlled by increased transcription of IQD12:
FIG. 3A shows the physical relationship between the two genomic constructs used to complement sun. Features of the genomic region are described in FIG. 1C. The 17.2 kb pHX2 construct contains IQD12, SDL1-like, HYP1 and nucleotides encoding the first 415 amino acids of HYP2 (residues 416-487 are deleted from this construct). The 14 kb pHX4 construct contains IQD12 and terminates 180 nucleotides upstream of the SDL1-like stop codon. Construct pHX2 extends to −1184 of the DELF1 methionine initiator codon, whereas pHX4 extends to −4407 of the DELF1 methionine initiator codon.
FIG. 3B shows that transformation of the genomic constructs into the round-fruited LA1589 genotype showed that IQD12 was expressed to higher levels in T1 plants transformed with pHX4 in comparison to lines transformed with pHX2. Total RNA was isolated from several fruits that were collected 5 dpa from the individual transformed plants. Northern blot filters were hybridized with 32P-labeled probes of IQD12 and eIF4α6 for normalizing of loading.
FIG. 3C shows that fruit shape index of T1 lines significantly correlates with IQD12 transcript levels (Pearson coefficient r=0.833, p<0.0001).
FIG. 3D shows that fruit shape index and IQD12 transcript levels of homozygous T2 plants derived from five independent pHX4 primary transformants. Two to five homozygous T2 plants were identified from selfed pHX4 primary transformants. Homozygosity was confirmed by progeny testing the T3 seed for the presence of the kanamycin resistance gene. The average fruit shape index (columns) and IQD12 transcript levels (line) were determined from the Northern blot data shown in FIG. 10B. Bars denote standard deviations. For comparison, four plants of each reciprocal NIL in the LA1589 background were included to determine IQD12 transcript level and fruit shape index comparisons to the T2 transgene families. Constitutive expression of IQD12 in lines that normally carry round fruit leads to extremely elongated fruit. Conversely, down regulation of IQD12 expression in lines that normally carry elongated fruit leads to round fruit. These results conclusively show that IQD12 is necessary and sufficient to regulate tomato fruit shape and therefore underlies SUN.
FIG. 3E shows that constitutive overexpression of IQD12 in the round-fruited LA1589 background results in extremely elongated fruit. Each fruit was taken from a plant that was independently transformed with the P35S:IQD12 construct. Fruit from the non-transformed round-fruited NIL (LA1589pp) is shown for comparison.
FIG. 3F shows that RNAi-mediated knock-down of IQD12 in the elongate-fruited NIL (LA1589ee) resulting in a significant reduction in fruit elongation. Each fruit represents an independent primary transformed RNAi line. The fruit from the non-transformed NIL (LA1589ee), is shown for comparison. Scale bar in (FIG. 3E) and (FIG. 3F) represents 1
FIGS. 4A-4B show that SUN is a member of the plant specific IQD family:
FIG. 4A shows that motifs predicted in SUN, IQD11 and AtIQD12 proteins. The entire IQD family was run through the MEME48 motif prediction program to identify conserved regions. Only the motifs with p<1.0e-5 are shown here. Motifs are numbered the same as previously reported26. Intron position conservation is indicated via broken connector lines.
FIG. 4B shows the phylogenetic relationships of SUN and its closest relatives. Bootstrapped phylogenetic tree (unrooted) of the IQ67 motif was generated after a complete sequence alignment using Clustal X (1.83) and the neighbor-joining method. The Arabidopsis proteins AtIQD11 and AtIQD12 were identified as At5G03960 and At5G13460, respectively. Other accessions were identified by their GenBank, SGN (Solanaceae Genomics Network), or JGI (“eugene3.00160363” (http://genome.jgi-psf.org/Poptr1—1/Poptr1—1.home.html)) numbers and a prefix denoting the genus and species. Br, Brassica rapa subsp. chinensis (bok choy); Gh, Gossypium hirsutum (upland cotton); Ha, Helianthus annuus (sunflower); Ls, Lactuca sativa (garden lettuce); Nt, Nicotiana tabacum (tobacco); Pt, Populus trichocarpa (black cottonwood); Sl, Solanum lycopersicum (tomato); St, Solanum tuberosum (potato); Vv, Vitis vinifera (vine grape). Numbers at nodes represent bootstrap values from 1000 trials. Values below 500 are not reported. IQD containing proteins that are closely related to SUN were not identified in monocots.
FIGS. 5A-5C show that the presence of the sun locus in other tomato varieties:
FIG. 5A shows that genetic analysis of segregation at sun in other cultivars. F2 populations were derived from crosses between LA1589 and the cultivars indicated above the lanes of the Southern blot shown in FIG. 5B. Via marker-assisted selection using marker cos103R (see FIG. 13—Table 4), 10 homozygous lycopersicum, 10 homozygous pimpinellifolium and 10 heterozygous seedlings were identified and grown to maturity. Average fruit shape index of eight fruit per plant was determined and significant association of shape and genotype was determined by Duncan's multiple range test at α of 0.01 and ANOVA. A “+” denotes significant association between fruit shape and genotype at sun; a “−” denotes the lack of association. The ovate allele was amplified from genomic DNA and analyzed as a dCAPS marker (FIG. 13—Table 4). A “+” denotes the presence of the pear-shaped allele of ovate; a “−” denotes the presence of the round allele of ovate (i.e. wild-type). The average fruit shape indices are shown below the marker scores and above the Southern blot lanes.
FIG. 5B shows that all S. lycopersicum lines that segregate at sun harbor the 4.3 kb EcoRV restriction fragment. The Southern blot was hybridized to a P32 labeled fragment that was amplified from phage clone EK36 using EP45 and EP46 primers (See FIG. 13—Table 4). This 4.3 kb EcoRV fragment signifies the presence of the duplication and hence sun. HG, Howard German; BL, Banana Legs; LJ, Long John; JD, Jersey Devil; Roma, RomaVF; YS, Yellow Stuffer; H1706, Heinz 1706.
FIG. 5C shows that the SUN gene was highly expressed in tomato varieties containing the transposed segment on chromosome 7. All S. lycopersicum varieties that harbor the 4.3 kb fragment express SUN to a high level, while expression is undetectable in the varieties without the transposed duplication.
FIGS. 6A-6E show a model for the segmental duplication and rearrangement at the sun locus:
FIG. 6A shows that the genome structure of the ancestral locus on chromosome 10. The nucleotide sequences in light green (GACCT) represent the target site duplication flanking Rider.
FIG. 6B shows that read-through transcription and template switch by Rider on chromosome 10. The red line and loop above the genome structure indicate the formation of the 24.3 kb retroelement RNA. The nucleotides in red (GCAGA) indicate the proposed site for the template switch. The sequence analysis of the sun locus on chromosome 7 reveals that the 24.3 kb segment is duplicated from chromosome 10 via a transposition-mediated event under the control of the autonomous LTR retroelement Rider. Transcription of Rider, read through and 3′ transduction, a template switch, followed by transposition repositions IQD12 farther away from the retroelement. The transposition positioned IQD12 in another genome context on chromosome 7, in the vicinity of the promoter of DEFL1, a gene whose function was abolished upon transposition.
FIG. 6C shows the formation of one large retroelement.
FIG. 6D shows the integration of Rider into chromosome 7 at the nucleotide site indicated in blue (ATATT).
FIG. 6E shows the structure of the sun locus on chromosome 7 featuring the target site duplication (blue-lettered nucleotides, ATATT). The arrows represent the direction of transcription of the genes. Dark gene arrows (HYP1, HYP2) indicate expressed and predicted functional genes, purple arrows (IQD12) indicate the rearranged gene, light green arrows indicate the retroelement Rider, yellow arrows (SDL1-like, DEFL1, last arrow on right) indicate pseudogenes. The red boxes numbered 1-3 indicate the identical Rider LTR.
FIGS. 7A-7C show the dosage-dependent expression of IQD12 and DEFL1 gene in LA1589 and Sun1642 Nearly Isogenic Lines (NILs):
FIG. 7A shows the Northern blot of RNA from NILs in the LA1589 background harboring 0, 1 or 2 copies of the Sun1642 allele of sun. The plant numbers above the lanes correspond to pedigree 06S559-6 for plant no “6” etc.
FIG. 7B shows the Northern blot of RNA from NILs in Sun1642 background harboring 0, 1 or 2 copies of the Sun1642 allele of sun. The plant numbers above the lanes correspond to pedigree 06S22-5 for plant no “5” etc.
FIG. 7C shows a graph showing the average relative transcript levels of IQD12 and DEFL1, normalized to eIF4α6 expression, in lines harboring 0, 1 or 2 copies of the Sun1642 allele of sun in the Sun1642 background. The data displayed in the graph are from the Northern blot shown in FIG. 9B. Error bar represents the standard deviation. The average mature stage fruit shape index is shown above the graph demonstrating that the degree of elongated fruit shape in the cultivated background is correlated to transcript levels of IQD12 and inversely correlated to transcript levels of DEFL1. Total RNA was isolated from 6-10 five days post anthesis (dpa) fruits of individual plants indicated above the lanes. The size-fractionated RNA was transferred to Hybond N membrane and hybridized sequentially to radioactivity-labeled tomato IQD12, DEFL1 and eIF4α6 probes. The denotion “ee” signifies two copies of the Sun1642 allele at sun; “pp” two copies of the LA1589 allele at sun; “ep” one copy of each parental allele.
FIGS. 8A-8C show the Northern blot of IQD12 expression in five pHX4 transgenic T1-derived T2 families. Expression analyses were performed on total RNA isolated from 6-10 fruit at five days post anthesis (dpa). Homozygous transgenic plants were identified from the selfed progenies of hemizygous pHX4 T1 lines indicated in FIG. 10—Table 1. The selection was based on signal intensity on a Southern blot and confirmed by examining the segregation of the kanamycin gene via PCR in the 48 T3 progenies from each of these T2 plants. For each blot shown, the transgenic plants and the NILs were grown simultaneously in the same greenhouse and tissues for RNA extraction were harvested at simultaneously as well.
FIG. 8A shows the Northern blot showing IQD12 expression in 5 dpa fruits of NILs in the LA1589 background compared to individual T2 plants derived pHX4 transgenic T1 lines. Plants were three months old when the fruits were harvested. The boxed plant numbers indicate the non-transgenic sibs of the T2 family. The non-transgenic T2 sibs show much lower IQD12 expression and a smaller fruit shape index, demonstrating that both increased IQD12 expression and fruit shape index is controlled by the pHX4 transgene. Most of the RNA from plants in the families 06S497 and 06S500 had degraded during extraction.
FIG. 8B shows the IQD12 expression in 5 dpa fruits of same plants as in FIG. 8A albeit at five months of age. The transcript levels obtained in FIG. 8B were used in the graph of FIG. 3D. The plant numbers marked by an asterisk (*) were excluded from the analysis because plant 9 of 06S496 and 22 of 06S501 were heterozygous whereas for plant number 8 of 06S500 the RNA had degraded during extraction. The fruit shape index for the plants displayed in FIGS. 8A and 8B was collected once.
FIG. 8C shows that cuttings of two transgenic families (06S497 and 06S500) were taken. The plants were three months after asexual propagation when 5 dpa fruits were harvested for RNA extraction. IQD12 expression was compared to that of the LA1589 NIL controls. The fruit shape index was recorded from these plants and shown below the lanes on the Northern blot. The results from these experiments convincingly showed that increase in expression of the transgenically introduced IQD12 resulted in increased fruit shape index which strongly suggested that this gene encodes SUN.
FIGS. 9A-9E show the IQD12 expression in P35S:IQD12 and RNAi:IQD12 transgenic lines:
FIG. 9A shows that constitutive expression of IQD12 leads to extremely elongated fruit in both the cultivated (Sun1642) and the wild type (LA1589) backgrounds. Round-fruited lines were transformed with P35S:IQD12. Total RNA was extracted from 5 dpa fruits except for lines 109-017, -019, -023 and -028. In the latter cases, anthesis-stage flowers were used instead because of irregular fruit set in these lines. The transgenic lines marked with an asterisk (*) (100-002 and 100-003), were obtained from P35S:IQD12 transformed into the round-fruited Sun1642 background, lacking the duplication. The fruit shape index for each line is shown below the Northern blot.
FIG. 9B shows that the elongated fruit shape and high expression of IQD12 under control of the CaMV 35S promoter is transmitted to next generation. Northern blot analysis was performed on total RNA isolated from anthesis-stage flowers of P35S:IQD12 T2 plants indicated above the lanes. To maintain these overexpressing lines which are essentially seedless, pollen of the primary transformants 109-017, -019, -023 and -028 were crossed to LA1589 pistils. ND, not determined. Boxed numbers indicate the non-transgenic sibs.
FIG. 9C shows the RNA knock down of IQD12 in the LA1589 NIL carrying the transposed segment, resulted in rounder fruit. Northern blot showing reduced transcript levels of IQD12 in 5 dpa fruit from each of the independent primary transgenic lines. Mature fruit shape index for each line was shown below the Northern blot.
FIG. 9D shows that the reduction of IQD12 transcript levels and fruit shape index is heritable to the T2 generation. The Northern blot shows IQD12 transcript levels in the non-transformed NILs (first 6 lanes; lines 07S27 and 07S26) compared to two transgenic families that knock down IQD12 transcript level. Total RNA was isolated from 5 dpa fruits of each plant. Boxed numbers indicate the non-transgenic sibs.
FIG. 9E shows a graph showing mature fruit shape index and IQD12 transcript level in the NILs compared to seven independent T2 families that down regulate IQD12 expression. The reduction of IQD12 transcript level led to a reduction in mature fruit shape index. Transcript levels of IQD12 in the transgenic plants was normalized to the transcript levels of eIF4α6 and expressed relative to that of the NIL carrying the Sun1642 allele of sun, which is set at “1”. A dashed line set at a fruit shape index value of 0.97 was drawn to facilitate the comparisons of the transgenic IQD12 knock down lines to that of the non-transformed NILs. Data for the Bars column was pooled from the 6 nontransgenic sib plants that segregated in the transgenic T2 progenies. The non-transgenic sibs came from three families (one from 07S21, one from 07S23 and four of 07S24).
FIG. 10 shows Table 1, listing the mature fruit shape index, IQD12 transcript levels and progeny testing of selected transformant in the round-fruited LA1589 background.
FIG. 11 shows Table 2, listing the mature fruit shape index of pHX2 and pHX4 primary transformants in the round-fruited Sun1642 background carrying the LA1589 allele of sun.
FIG. 12 shows Table 3, listing the fruit shape index of plants that over or under express IQD12.
FIG. 13 shows Table 4, listing the primers [SEQ ID NOS: 14-14], respectively in order of appearance, as used in the examples described herein.
FIGS. 14A-14C show expression of SUN in different tissues:
FIG. 14A shows the gene structure at the sun locus. The 24.7 kb duplication, including SUN, disrupted the expression of DEFL1.
FIG. 14B shows the expression of SUN and DEFL1 in floral tissues. Northern blot containing RNA isolated from floral organs at the time of anthesis. The blot was sequentially probed with SUN, DEFL1 and lastly with eIF4α-6 as loading control. Se, sepal; Pe, petal; St, stamen; Ov, ovary. Tissues were isolated from the LA1589 and Sun1642 NILs that differ at the sun locus. ee, containing an extra copy of SUN; pp, lacking the extra copy of SUN.
FIG. 14C shows the expression of SUN and DEFL1 in different tissues. Tissues were isolated from the LA1589 and Sun1642 NILs that differ at the sun locus. ee, containing an extra copy of SUN; pp, lacking the extra copy of SUN. R, root; H, hypocotyl; C, cotelydon; L, leaf; S, shoot apex. In lines carrying the duplication, SUN is highly expressed in sepals, ovaries, hypocotyl and shoot apex. The ancestral SUN gene is expressed in roots and at low levels in other tissues. DEFL1 is expressed in the same tissues as the duplicated copy of SUN in plants that lack the 24.7 kb duplication (pp, lacking an extra copy of SUN). This indicates that the promoter of DEFL1 is driving expression of the duplicated copy of SUN.
FIGS. 15A-15C: Fruit shape index and SUN expression changes during tomato flower and fruit development:
FIG. 15A shows the fruit shape index (length/width ratio) as a function of the number of days post anthesis. Fruit shape changes overlay the fruit/seed developmental landmarks indicated above the graph. The black triangles represent the fruit shape indices of the near isogenic lines (NILs) carrying two copies of the SUN gene whereas the grey circles represent the NILs with only one copy of SUN. The largest difference in fruit shape index is achieved at fruit landmark 3 and 4, coinciding with the landmarks 4-16 cell and globular stage of the embryo. Fruit shape index were collected from three inflorescences per plants of five for each genotype. Data shown are mean±standard error.
FIG. 15B shows SUN expression in the developing fruits of LA1589 NILs. Northern blots were performed on LA1589ee (carrying two copies of the SUN gene) and LA1589pp (carrying one copy of SUN). Total RNA was extracted from pooled tissues of five plants per genotype and hybridizations were conducted with SUN as probe. SUN expression is very high starting at anthesis until 20 days post anthesis. Its expression has dropped dramatically 25 days post anthesis which is prior to the fruit ripening and seed germination stage.
FIG. 15C shows SUN expression in floral buds of LA1589 NILs. Northern blots were performed on total RNA isolated from entire flowers or buds at the times indicated above the lanes (in days). The “0” timepoint denotes anthesis, the other values indicate days prior (−) or post (+) anthesis. SUN's expression is low 2 days pre-anthesis but increases dramatically until 2 days post pollination in the lines carrying two copies of SUN (LA1589ee). The increase in SUN expression precedes the change in fruit shape index shown in FIG. 15A. In the lines carrying only one copy of SUN (LA1589pp), SUN expression is low, however DEFL1 expression in LA1589pp follows a similar kinetic as SUN expression in the LA1589ee indicating that the DEFL1 promoter drives SUN expression (see also FIG. 15).
FIGS. 16A-16B: The effect of SUN on leaflet shape:
FIG. 16A shows the leaflets of cultivated tomato.
FIG. 16B shows leaflets of the wild relative S. pimpinellifolium accession LA1589. The leaves shown are from plants without the extra copy of SUN (pp), with the extra copy of SUN (ee) or SUN expressed under the constitutive 35S promoter (ox). The most notable feature is the pointed shape of the leaf and increased serrated margins when SUN is expressed (compare pp and ee). These features are accentuated when SUN is overexpressed (compare ee and ox). These results indicate that in addition to fruit shape, leaf shape is dramatically altered as well.
FIGS. 17A-17F shows the effect of overexpression of SUN on ovary, fruit and compound leaf shape in tomato:
FIGS. 17A-17C show the S. pimpinellifolium LA1589 background. Ovary shape of anthesis-stage flowers (FIG. 17A), fruit shape (FIG. 17B) and compound leaf shape (FIG. 17C) of wild type (LA1589pp) and 5 independent transformants. Note the very elongated slender shape of the ovary, fruit and the twisted shape of the leaves.
FIGS. 17D-17F show the S. lycopersicum Sun1642 background. Ovary shape of anthesis-stage flowers (FIG. 17D), fruit shape (FIG. 17E) and compound leaf shape (FIG. 17F) of wild type (Sun1642pp) and several independent transformants. When SUN is overexpressed under control of the 35S promoter, the fruit shape is already determined at the time of anthesis. Moreover, leaflets and compound leaf shape is also greatly affected when SUN is overexpressed. The leaves are twisted and the leaflets are more pointed in shape.
FIGS. 18A-18C show the cell size and number differences in the longitudinal direction of LA1589 NIL fruit differing at sun.
FIG. 18A shows the length of different parts of the fruit at 5 days post anthesis. All fruit parts are more elongated in the presence of SUN.
FIG. 18B shows the cell size is only significantly different in the distal end of the fruit.
FIG. 18C shows the ratio of fruit length and cell size shows that the septum and proximal end of the fruit have significantly more cells.
FIGS. 19A-19C show the cell size and number differences in the latitudinal direction of LA1589 NIL that differ at sun:
FIG. 19A shows the width of the fruit at 5 days post pollination. Total fruit and septum width are significantly smaller in the NILs carrying the SUN duplication.
FIG. 19B shows the cell number in the septum is significantly lower in the NILs carrying the SUN duplication.
FIG. 19C shows the cell size is not significantly different in the septum or pericarp. The results shown in FIGS. 18 and 19 demonstrate that SUN controls directional cell division predominantly in the septum and proximal end of the fruit. High expression of SUN leads to increased cell division in the longitudinal direction and reduced cell division in the latitudinal direction. This suggests that SUN can affect the shape of any organ in any plant species depending on where and when the gene is expressed.
FIG. 20 shows the stem structure of S. lycopersicum cv Sun1642pp (without the SUN duplication and carrying round fruit) and Sun1642pp overexpressing SUN.
FIG. 21 shows Table 5 shows the near isogenic lines that differ at sun in both the Sun1642 and the LA1589 background show changes in fruit shape index, leaflet shape index (see also FIG. 16), sepal and ovary shape index and to a lesser extent petal shape index, and seed weight. Fruit weight, number of seed per fruit, hypocotyl and internode length is not altered. The fact that SUN does not affect fruit weight but only the shape strongly indicates that the gene acts to redirect growth without increasing growth. Again, this finding shows that SUN may be able to alter direction of growth of any plant organ.
FIG. 22 shows Table 6 shows that leaf shape, fruit shape, seed number per fruit, seed and fruit weight are similar in the line expressing SUN under its own promoter compared to the NIL carrying the SUN gene duplication. This shows that only SUN but neither DEFL1 nor one of the HYP genes (hypothetical, see FIG. 14) affect shape of plant organs and seed weight.



The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.
The GenBank accession number EF094939 [SEQ ID NO:1] for nucleotide sequence: file BAC—72D08_Lycopersicum_es, corresponds to the sequence on chromosome 10.
The GenBank accession number EF094940 [SEQ ID NO:2] for nucleotide sequence: file sunLesc_Lycopersicu_escul corresponds to the entire sequence of the sun locus in some cultivated tomato (the varieties that carry the duplication).
SUN Exon/Intron Positions in EF094940 [SEQ ID No:2] are as follows















EXON
Genomic coordinates
mRNA coordinates









Exon 1
13386-13522
 1-137



Intron 1
13523-13964



Exon 2
13965-14051
138-224



Intron 2
14052-14135



Exon 3
14136-14447
225-536



Intron 3
14448-15013



Exon 4
15014-15238
537-761



Intron 4
15239-15386



Exon 5
15387-15518
762-893



Intron 5
15519-15685



Exon 6
15686-16858
 894-2066










The GenBank accession number EF094941 [SEQ ID NO:3] for nucleotide sequence: file sunLpip_Lycopersicum_pimp corresponds to the sequence of the sun locus in the wild species.
The SUN gene amino acid sequence [SEQ ID NO:4]:







MGKRRNWFTFVKRLFIPETESTADQKKPKRWRCCFLRKFKLRKCPAITSA


 


PQQTLPEAKGTPQQTLTEAKEQQRKHAFAVAIATAAAAEAAVAAANAAAD


 


VIRLTDAPSEFKRKRKQAAIRIQSAYRAHLAQKALRALKGVVKLQAVIRG


 


EIVRGRLIAKLKFMLPLHQKSKTRVNQIRVPTFEDHHDKKLINSPREIMK


 


AKELKLKCKSLSTWNFNLASEQDSEALWSRREEAIDKREHLMKYSFSHRE


 


RRNDQTLQDLLNRKQNRRSYRIDQLVELDAPRKAGLLEKLRSFTDSNVPL


 


TDMDGMTQLQVRKMHRSDCIEDLHSPSSLPRRSFSNAKRKSNVDDNSLPS


 


SPIFPTYMAATESAKAKTRSNSTAKQHLRLHETLSGQHSPYNLKISSWRL


 


SNGEMYDSARTSRTSSSYMLI






The DNA sequence listing the clone that, when transformed into plants resulted in elongated fruit. The DNA sequence is shown in EU491503_suncdna [SEQ ID No:5] SunLesc.txt/genbank [SEQ ID Nos:4, 7, 8, 9, 10, 10, 11, 12, 13].
The construct that is encompassed by pHX4 (EK60): nt 7305-21371 [SEQ ID No:6], when transformed into tomato plants, confers an elongated fruit phenotype. The regulatory element for elevated transcription is located from 7305 to 10528 nt. The entire promoter of SUN spans from 7305-13386. The coding region of SUN starts in exon 2 at nt 13974. The 3′ UTR of SUN starts in exon 6 at nt 16204. The 3′ downstream region of the SUN spans from nt 16858-21371.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
The principles and operation of the present invention may be better understood with reference to the accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
In an important aspect, the present invention relates to polynucleotides and polypeptides, for example, for modifying phenotypes of plants. Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses, for example. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of “incorporation by reference” is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the invention.
Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used and the manufacture or laboratory procedures described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. Where a term is provided in the singular, it is also contemplated that aspects of the invention described by the plural of that term. The nomenclature used and the laboratory procedures described below are those well known and commonly employed in the art. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given. Other technical terms used have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries. The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” includes a plurality of such plants, and a reference to “a stress” is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.
An “isolated polypeptide,” whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.
“Homology” refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.
“Hybridization complex” refers to a complex between two nucleic acid molecules by virtue of the formation of hydrogen bonds between purines and pyrimidines.
“Identity” or “similarity” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases “percent identity” and “% identity” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences.
“Sequence similarity” refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.
An “IQ motif” is defined as an amino acid sequence of 20-40 amino acids in length containing an isoleucine residue (designated “I”) immediately followed by a glutamine residue (designated “Q”) which has at least 50% sequence similarity to the consensus sequence.
“Alignment” refers to a number of DNA or amino acid sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues) may be readily and graphically identified. The number of components in common is related to the homology or identity between the sequences. Alignments may be used to identify “conserved domains” and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art.
A “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences.
A “conserved domain”, with respect to presently disclosed polypeptides refers to a domain that exhibits a higher degree of sequence homology, such as at least 26% sequence similarity, at least 16% sequence identity, preferably at least 40% sequence identity, preferably at least 65% sequence identity including conservative substitutions, and more preferably at least 80% sequence identity, and even more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% amino acid residue sequence identity of a polypeptide of consecutive amino acid residues. It is to be understood that ranges within these percentages is also within the contemplated scope of the disclosure herein.
A fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be “outside a conserved domain” if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
“Complementary” refers to the natural hydrogen bonding by base pairing between purines and pyrimidines. For example, the sequence A-C-G-T (5′→3′) forms hydrogen bonds with its complements A-C-G-T (5′→3′) or A-C-G-U (5′→3′). Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or “completely complementary” if all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of the hybridization and amplification reactions.
“Fully complementary” refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, and the two sequences have the same number of nucleotides.
The terms “highly stringent” or “highly stringent condition” refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are known to those skilled in the art.
In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure. The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate sequences having similarity to sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed sequences, such as, for example, sequences having 60% identity, or more preferably greater than about 70% identity, most preferably 72% or greater identity with disclosed sequences.
The term “variant”, as used herein, may refer to polynucleotides or polypeptides that differ from the presently disclosed polynucleotides or polypeptides, respectively, in sequence from each other, and as set forth herein.
With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.
Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent polypeptide. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the polypeptide and homolog polypeptides of the invention. A polypeptide sequence variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the functional or biological activity of the polypeptide is retained.
The term “plant” includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, and the like), and progeny of same. A plant also refers to plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
A “transgenic plant” refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant.
“Control plant” refers to a plant that serves as a standard of comparison for testing the results of a treatment or genetic alteration, or the degree of altered expression of a gene or gene product. Examples of control plants include plants that are untreated, or genetically unaltered (i.e., wild-type).
“Wild type”, as used herein, refers to a cell, tissue or plant that has not been genetically modified to knock out or overexpress one or more of the presently disclosed transcription factors. Wild-type cells, tissue or plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants in which expression is altered or ectopically expressed, e.g., in that it has been knocked out or overexpressed.
The term “growing” or “regeneration” as used herein means growing a whole plant from a plant cell, a group of plant cells, a plant part (including seeds), or a plant piece (e.g., from a protoplast, callus, or tissue part).
A “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some non-limiting instances, this characteristic is visible to the human eye, such as seed, plant size or fruit or vegetable shape, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring uptake of carbon dioxide, altered expression or suppression of a hormone, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.
“Trait modification” refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease in an observed trait (difference), at least a 5% difference, at least about a 10% difference, at least about a 20% difference, at least about a 30%, at least about a 50%, at least about a 70%, or at least about a 100%, or an even greater difference compared with a wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution of the trait in the plants compared with the distribution observed in wild-type plants.
The term “transcript profile” refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular gene in a suspension cell is the expression levels of a set of genes in a cell overexpressing that gene compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that gene. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods.
“Altered expression” in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term “altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.
The term “overexpression” as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more polypeptides are under the control of a strong expression signal, such as one of the promoters described herein (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may occur throughout a plant or in specific tissues of the plant, depending on the promoter used.
Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present polypeptides. Overexpression may also occur in plant cells where endogenous expression of the present polypeptides or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or “overproduction” of the polypeptide in the plant, cell or tissue.
Similarly, the term “under expression” as used herein refers to a lesser expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Under expression can occur when, for example, the genes encoding one or more polypeptides are under the control of a strong expression signal, such as one of the promoters described herein (e.g., the cauliflower mosaic virus 35S transcription initiation region). Under expression may occur throughout a plant or in specific tissues of the plant, depending on the promoter used.
Under expression may take place in plant cells normally having expression of polypeptides functionally equivalent or identical to the present polypeptides. Under expression may also occur in plant cells where endogenous expression of the present transcription factors or functionally equivalent molecules normally occurs, but such normal expression is at a higher level. Under expression thus results in a lesser than normal production, or “under” of the polypeptides in the plant, cell or tissue.
Thus, in a first broad aspect, there is provided herein a chromosome 7 region, which region, when introduced into a cultivated plant genetic background alters at least one trait in the plant.
Non-limiting examples of plants for which one or more traits can be altered include gymnosperms, angiosperms and mosses.
Non-limiting examples include including monocots and dicots, such as crop plants, ornamental plants, and non-domesticated or wild plants. Further examples include plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood- or pulp-producing trees, vegetable plants, fruit plants, and ornamental plants. Non-limiting examples of plants of interest include grain crop plants (such as wheat, oat, barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicots including alfalfa, vetch, clover, and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee; wood- or pulp-producing trees; vegetable crop plants such as legumes (for example, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, the brassicas (for example, cabbages, kales, mustards, and other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example, cucumbers, melons, summer squashes, winter squashes), edible alliums (for example, onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers, groundcherries), and edible members of the Chenopodiaceae (for example, beet, chard, spinach, quinoa, amaranth); fruit crop plants such as apple, pear, citrus fruits (for example, orange, lime, lemon, grapefruit, and others), stone fruits (for example, apricot, peach, plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado, and berries; and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental groundcovers, and ornamental grasses. Further examples of dicot plants include, but are not limited to, canola, cotton, potato, quinoa, amaranth, buckwheat, safflower, soybean, sugarbeet, and sunflower, more preferably soybean, canola, and cotton. Further examples of monocots include, but are not limited to, wheat, oat, barley, maize, rye, triticale, rice, ornamental and forage grasses, sorghum, millet, and sugarcane, more preferably maize, wheat, and rice.
Non-limiting examples of suitable members of the Solanaceae family include tomato plants that may have a range of genotypes used in the production of commercial tomato varieties. Suitable plants can be used for processing into tomato paste, dice and whole peel production, fresh market tomatoes for open field production, staked cultivation in the open field and protected cultivation such as in a glasshouse.
In one particular aspect, there is provided herein a portion of a targeted site duplication into chromosome 7 resulting in elongated-fruited tomato. This duplicated chromosome 7 fragment which spans from a region between tomato markers Lp81B9-F and Lp61O2-R is capable of, when introduced into the genetic background of a cultivated tomato, altering the fruit shape index of fruits produced thereby, while at the same time maintaining the desired phenotypic traits of the cultivated plant.
Thus, in another aspect, there is provided cultivated tomato plants producing fruits and seeds, such as, for example, hybrid seeds, of commercial value, which plants are generated according to the teachings of the present invention by introducing a chromosomal region associated with a high fruit shape index, into a genetic background of a cultivated tomato.
In a first aspect, there is are provided herein isolated polynucleotides, including: (a) a sequence encoding a polypeptide, wherein the sequence is at least one of SEQ ID NOs: 1, 2, 3, 4 and 5, or segments thereof; (b) a variant of any of the sequences of (a) or (b) that has at least 70% sequence identity to a sequence of (a); (c) an orthologous sequence of any of the sequences of (a) or (b) that has at least 70% identity to a sequence of (a); (d) a paralogous sequence of any of the sequences of (a) or (b) that has at least 70% identity to a sequence of (a) or a paralogous sequence with 70% identity to the IQ motif thereof; (e) a sequence that hybridizes to any of the sequence of (a) under stringent conditions; and (f) a sequence encoding a polypeptide comprising a conserved domain that has at least 70% sequence homology with a conserved domain of a polypeptide encoded by any of the sequences of (a)-(e), wherein the conserved domain is required for the function of the polypeptide encoded by any of the sequences of (a)-(e) in regulating expression of the nucleotide and altering a trait in a transgenic plant.
In on embodiment, wherein the recombinant polynucleotide is operably linked to at least one regulatory element being effective in controlling expression of the recombinant polynucleotide when the recombinant polynucleotide is transformed into a plant. In certain embodiments, the polynucleotide is incorporated within an expression vector.
Also, in certain embodiments, there is provided herein a polynucleotide comprising one or more constitutive, inducible, or tissue-specific promoters operably linked to the polynucleotide sequence as described above. In certain embodiments, the expression vector is incorporated into a cultured host cell.
In another aspect, there is provided herein a transgenic plant that expresses at least one polynucleotide described herein, where at least a part of the transgenic plant has an altered trait as compared to a non-transgenic plant or wild-type plant.
In certain embodiments, the altered trait is one or more of: sensitivity to hormone levels, altered shape of at least part of the plant, altered plant size; altered leaf shape; altered vegetable shape, altered fruit shape; at least partially parthenocarpic fruit, increased SUN levels, and decreased SUN levels. Also, in certain embodiments, the altered trait is an overexpression of at least a portion of one of the isolated polynucleotides wherein the altered trait comprised a parthenocarpic fruit. Also, in certain embodiments, the altered trait is an expression of at least a portion of one of the isolated polynucleotides wherein the altered trait comprises an elongated fruit shape.
In a further aspect, there is provided herein transgenic plants where the plant is a plant that expresses one or more proteins from the IQD family of proteins. Such transgenic plants can be selected from one or more of: gymnosperms, angiosperms and mosses.
In certain embodiments, the plant is selected from one or more of: monocots and dicots, including, but not limited to crop plants, ornamental plants, and non-domesticated or wild plants.
In certain embodiments, the transgenic plant is selected from the Solanaceae family. In certain embodiments, the transgenic plant is a tomato plant. In certain embodiments, the transgenic plant is a tomato plant near isogenic line as described herein.
In another aspect, there is provided herein a method for producing a transgenic plant having an altered trait as compared to a non-transgenic or wild-type plant where the method includes: (a) providing an expression vector comprising: (i) a polynucleotide as described herein; and (ii) at least one regulatory element flanking the polynucleotide sequence, the at lest one regulatory element being effective in controlling expression of the recombinant polynucleotide in a target plant; (b) introducing the expression vector into a plant cell, thereby producing a transgenic plant cell; (c) growing the transgenic plant cell into a transgenic plant and allowing the transgenic plant to express or suppress a polypeptide encoded by the polynucleotide, the polypeptide having the property of altering a trait in a plant as compared to a non-transgenic plant that does not express or suppress the polypeptide; and (d) identifying at least one transgenic plant with an altered trait by comparing the transgenic plant with the non-transgenic plant.
In certain embodiments, the method further includes: (e) selfing or crossing the at least one transgenic plant with an altered trait with itself or another plant, respectively; and (f) growing a progeny plant from seed that develops as a result of the selfing or crossing, thus producing a transgenic progeny plant having an altered trait.
In another aspect there is provided herein a cell transformed with at least one polynucleotide described herein. In certain embodiments, the cell is a plant cell. Also provided herein is plant or plant tissue grown from such plant cell.
In another aspect, there is provided herein a transformed or transgenic plant, plant part, plant seed, plant cell, or the transgenic progeny thereof, comprising a sequence encoding a polypeptide, wherein the nucleotide sequence is at least one of: SEQ ID NO: 1, 2, 3, 4 and 6, and combinations thereof. In certain embodiments, the transgenic plant is selected from the Solanaceae family, such as, but not limited to a tomato plant. In certain embodiments, the plant is a tomato plant near isogenic line as described herein.
In another aspect, there is provided herein a transformed plant comprising in its genome at least one stably incorporated nucleotide construct comprising a promoter that drives expression in a plant operably linked to at least on of the isolated polypeptides described herein.
In another aspect, there is provided herein a mRNA molecule flanked by part of two LTRs, the R segment and U5 region at the 5′ end of the mRNA and the R segment and U3 region at the 3′ end of the mRNA, substantially as shown in FIG. 6C. In another aspect, there is provided herein a molecule where the LTR1 and LTR3 are on chromosome 7 and flank an entire duplicated fragment, and immediately flanking LTR1 and LTR3 are a 5 bp motifs “ATATT”.
In another aspect, there is provided herein a clone, pHX4, comprising a full length SUN gene.
In another aspect, there is provided herein a plant transformed with the pHX4 clone.
In another aspect, there is provided herein a clone further including a cis-element located in a 3.2 kb region upstream of DEFL1.
In another aspect, there is provided herein a method for altering at least one trait of a plant, comprising causing a duplication event mediated by an autonomous Long Terminal Repeat retroelement, wherein the transposition of the retroelement results in the placement of a SUN gene proximal to a regulatory sequence of another gene results in an altered expression compared to its paralog at an ancestral location. In certain embodiments, a gain-of-function mutation results from the transposition event mediated by the autonomous LTR-retroelement, and wherein the transposition of the retroelement is associated with 3′ transduction of nearby genes as well as a second rearrangement that moves the SUN gene from upstream of the retroelement to 20 kb downstream of the retroelement.
In another aspect, there is provided herein a method for making a plant having at least one fruit having a shape that is different from a naturally occurring fruit, comprising transforming a SUN inverted repeat construct into a near isogenic line of the fruit, and growing the plant.
In another aspect, there is provided herein a method of producing a fruit, comprising: a) growing a plant having at least one polypeptide described herein to produce a fruit, and b) harvesting the fruit.
In another aspect, there is provided herein a method of vegetatively propagating a plant comprising: collecting part of a plant grown; and obtaining a plantlet from the part. The method can further comprise growing a plant from the plantlet. Also, in certain embodiments, method can further include harvesting a fruit from the plant grown from the plantlet.
In another aspect, there is provided herein food and food products comprising the fruit of the plants described herein.
In another aspect, there is provided herein a near isogenic line (NIL) comprising a Sun1642 background.
In another aspect, there is provided herein a near isogenic line (NIL) comprising a LA1589 background.
In another aspect, there is provided herein a 17.2 kb pHX2 construct containing IQD12, SDL1-like, HYP1 and nucleotides encoding the first 415 amino acids of HYP2.
In another aspect, there is provided herein a 14 kb pHX4 construct containing IQD12 and terminating 180 nucleotides upstream of the SDL1-like stop codon.
In another aspect, there is provided herein a transformation construct made by subcloning the entire phage subclone insert of plasmids pEK59 and pEK60, respectively (released by NotI and blunted-ended using Klenow) into the Klenow-blunted ended BamHI-digested binary vector pCIB10G. In certain embodiments, the transformation construct comprises pHX2. In certain embodiments, the transformation construct comprises pHX4.
In another aspect, there is provided herein a RNAi:IQD12 construct, pHX8, generated by cloning 512 bp fragments of the IQD12 cDNA (from nucleotide 16,154 to 16,646 of the genomic sequence EF094940 [SEQ ID NO:2]), amplified using primer EP527 and EP528 from reverse transcribed mRNA, in the sense and antisense directions into pFGC5941.
In another aspect, there is provided herein a method for over expressing IQD12, a 1.4 kb fragment of IQD12 cDNA (corresponding to nucleotide 13,460-16,280 of the genomic sequence EF094940 [SEQ ID NO:2]), comprising: amplifying from reverse transcribed mRNA using primers EP519 and EP520 and subcloning between a CaMV 35S RNA promoter and NOS terminator of pCIB710.
In another aspect, there is provided herein a method for probe labeling expressing IQD12, a 1.4 kb fragment of IQD12 cDNA (corresponding to nucleotide 13,460-16,280 of the genomic sequence EF094940 [SEQ ID NO:2], comprising amplifying from reverse transcribed mRNA using primers CME5F and CME5R.
In another aspect, there is provided herein a method for silencing expressing IQD12, a 1.4 kb fragment of IQD12 cDNA (corresponding to nucleotide 13,460-16,280 of the genomic sequence EF094940 [SEQ ID NO:2]), comprising amplifying from reverse transcribed mRNA using primers EP527 and EP528.
In another aspect, there is provided herein a construct comprising plasmid pHX8.
In another aspect, there is provided herein a plasmid construct pHX8 transformed into near isogenic line (NIL) carrying a Sun1642 allele in a LA1589 background.
In another aspect, there is provided herein a construct comprising plasmid pHX2.
In another aspect, there is provided herein a plasmid construct pHX2 useful to transform round-fruited NIL plants in both the LA1589 and Sun1642 backgrounds.
In another aspect, there is provided herein a construct comprising plasmid pHX4.
In another aspect, there is provided herein a plasmid constructs pHX4 useful to transform round-fruited NIL plants in both the LA1589 and Sun1642 backgrounds.
In another aspect, there is provided herein a construct comprising plasmid pEK69.
In another aspect, there is provided herein a plasmid construct pEK69 useful to transform round-fruited NIL plants in both the LA1589 and Sun1642 backgrounds.
In another aspect, there is provided herein a near isogenic line (NIL) comprising a plant differing at sun constructed in Sun1642 background or LA1589 backgrounds, wherein the plant is made by sequential backcrosses to a recurrent parent using marker-assisted selection.
In another aspect, there is provided herein a 6.08 kb upstream region DEFL1 used as a promoter.
In another aspect, there is provided herein a vector comprising at least one of the polypeptides described herein. In another aspect, there is provided herein a plant cell transformed with such vector. In another aspect, there is provided herein a plant transformant comprising such plant cell. In another aspect, the plant transformant is tomato. In another aspect, there is provided herein a progeny or a clone of such plant transformant.
In another aspect, there is provided herein a method for producing a plant, comprising introducing at least one of the polypeptides described herein into a plant cell, and regenerating a plant transformant from the plant cell.
In another aspect, there is provided herein a method for altering at least one of a leaf and fruit shape of a plant, comprising introducing and expressing the polypeptide in the plant wherein the expressing the polypeptide alters the shape of the leaf and/or fruit, as compared to a plant that does not express at least one of the polypeptides described herein.
In another aspect, there is provided herein an isolated host cell transformed with a vector comprising at least one polypeptide described herein.
In another aspect, there is provided herein a process for altering at least one trait of a plant, or part thereof, comprising increasing SUN activity in the plant, or part thereof. In certain embodiments, the SUN has the amino acid sequence of SEQ ID NO:4 or an amino acid sequence with at least 60% sequence homology to SEQ ID NO:4 and which has SUN activity. In certain embodiments, the process includes introducing into the plant, or part thereof, a mutation into the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:4 or into a polynucleotide sequence encoding an amino acid sequence with at least 70% sequence homology to SEQ ID NO:4 and which has SUN activity.
In certain embodiments, the process includes introducing into the genome of the plant, or part thereof, in a sense or antisense orientation, a polynucleotide sequence of SEQ ID NO:4 or a polynucleotide sequence with at least 70% sequence homology to SEQ ID NO:4, wherein the homologous polynucleotide sequence inhibits SUN activity.
In another aspect, there is provided herein an isolated polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:4 or encoding an amino acid sequence with at least 95% sequence homology with the amino acid sequence of SEQ ID NO:4. In another aspect, there is provided herein a vector comprising such polynucleotide.
In another aspect, there is provided herein an isolated polypeptide involved in the determination of fruit shape of plants, wherein the polypeptide is selected from one or more of: (a) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO:4; (b) a DNA comprising a coding region of the nucleotide sequence of SEQ ID NO:2; (c) a DNA encoding a protein comprising an amino acid sequence with one or more amino acid substitutions, deletions, additions, and/or insertions in the amino acid sequence of SEQ ID NO:4; and (d) a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO:2.
In another aspect, there is provided herein an isolated polypeptide encoding a partial peptide of a protein comprising at least one of the sequence of SEQ ID NO:4 or 6.
In another aspect, there is provided herein an isolated polypeptide comprising a promoter region of the nucleotide sequence of SEQ ID: NO:2. In another aspect, there is provided herein a vector comprising such polypeptides having one or more of SEQ ID NOS: 2, 4 or 6. In another aspect, there is provided herein a host cell carrying such vector. In another aspect, there is provided herein a plant cell carrying such vector. In another aspect, there is provided herein a plant transformant comprising such plant cell. In another aspect, there is provided herein a plant transformant that is a progeny or a clone of such plant transformant. In another aspect, there is provided herein a propagation material of such plant transformant.
In another aspect, there is provided herein a method for producing a plant transformant, wherein the method comprises the steps of introducing at least one polypeptide described herein into a plant cell, and regenerating a plant from the plant cell.
In another aspect, there is provided herein an isolated polynucleotide comprising at least 15 continuous nucleotides that are complementary to the nucleotide sequence of SEQ ID NO:2, or a sequence complementary thereto.
In another aspect, there is provided herein a method for increasing the regeneration ability of a plant, wherein the method comprises the step of expressing at least one polypeptide described herein in a cell of a plant.
In another aspect, there is provided herein an agent for altering at least one trait of a plant, wherein the agent comprises at least one polypeptide described herein, or a vector thereof as an active ingredient.
In another aspect, there is provided herein a method for determining the ability of a plant cell to produce a fruit with an altered shape, wherein the method comprises detecting the expression of at least one polypeptide described herein or a protein expressed thereby in the plant cell.
In another aspect, there is provided herein a method for determining the ability of a plant cell to produce a fruit with an altered shape, comprising detecting the expression of the polypeptide in the plant cell.
In another aspect, there is provided herein a method for improving the ability of a plant to produce a fruit with an altered shape, comprising regulating the activity of at least one protein produced by expression of at least one polypeptide described herein in the plant.
In another aspect, there is provided herein a method for selecting a transformed plant cell, comprising: (a) introducing a plant cell with a vector comprising at least one polypeptide described herein as a selection marker; (b) culturing the plant cells; and, (c) selecting plant cells that have acquired regeneration ability.
In another aspect, there is provided herein a method for altering the ability of a plant to produce a fruit with an altered shape, comprising substituting an endogenous polypeptide in a plant by crossing.
In another aspect, there is provided herein a plant cell transformed by an expression vector comprising an isolated molecule in antisense orientation, wherein expression of the vector in the plant cell results in an altered fruit shape index as compared to a corresponding wild-type plant, and wherein the molecule comprises (a) the sequence shown in SEQ ID NOS: 1, 2, 3, 4 or 6, or variants thereof, or (b) a sequence encoding the same sequence as the sequence of (a), but which is degenerate in accordance with the degeneracy of the genetic code. In certain embodiments, the molecule is SUN, SEQ ID NO:4.
In another aspect, there is provided herein a seed produced by a transgenic plant comprising the plant cell, where the seed is true breeding for an altered fruit shape index of a daughter plant as compared to a wild-type variety of plant cell.
In another aspect, there is provided herein a recombinant antisense expression vector comprising: (a) a promoter functional in a plant cell; and (b) an isolated molecule comprising SUN, SEQ ID NO: 4, wherein the molecule is operably linked in antisense orientation to a promoter. In another aspect, there is provided herein a method for producing a transgenic plant having an altered fruit shape as compared to the corresponding wild-type plant, comprising: (a) transforming plant cells by introducing the recombinant antisense expression vector; (b) producing plants from the transformed cells and (c) selecting a whole plant exhibiting an altered fruit shape index.
In another aspect, there is provided herein a method for altering the size of the fruit of a plant, comprising: (a) introducing the recombinant antisense expression vector into a plant cell; (b) regenerating the plant cell into a transgenic plant; (c) evaluating the whole plant for an altered fruit shape by comparing the plant with the introduced expression vector to a corresponding wild-type plant. In certain embodiments, the transgenic plant exhibits increased fruit shape index growth as compared to the corresponding wild-type plant.
In another aspect, there is provided herein a plant cell transformed with an isolated amino acid sequence in antisense orientation, wherein the amino acid sequence is SUN, SEQ ID NO:4, or the complement thereof, or a molecule encoding the same amino acid sequence as SEQ ID NO:4, but which is degenerate in accordance with the degeneracy of the genetic code, wherein expression of the sequence in the plant cell results in an altered fruit shape of a resulting plant as compared to a corresponding wild-type plant.
In another aspect, there is provided herein a transgenic plant comprising the plant cell as described herein.
In another aspect, there is provided herein a seed produced by the transgenic plant described herein where the seed comprises the isolated nucleotide sequence in antisense orientation.
In a particular embodiment, the sequence, as describe herein may be from any tomato species, particularly the S. lycopersicum fruit tomato plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the invention may also include fragments of the present amino acid sequences.
EXAMPLES
According to one particular aspect, there is provided herein a cultivated tomato plant having a genome including an altered expressing through duplication and reposition of a gene derived from an elongated fruit tomato line. The elongated-fruited tomato is characterized by fruits having a high fruit shape index, i.e., where the fruit's height is greater that its width. The duplication/reposition gene, identified herein as IQD12 and/or SUN, includes a portion of chromosome 10 present as a duplicated fragment on chromosome 7. The SUN gene is responsible for a desired fruit elongation trait as compared to a wild-type tomato plant.
According to another aspect, there is provided herein a method of generating a tomato plant having fruits characterized by an increased fruit shape index, the method comprising the step of introducing to a genome of the tomato plant a construct derived from a elongated-fruited tomato, the construct including a portion of chromosome 10 of the elongated-fruited tomato duplicated on the chromosome 7.
As such, an evidence of the altered fruit shape index property may come by an introduction thereof into any cultivated variety of a construct that includes at least the SUN gene, as if further exemplified and described in the Examples section which follows.
Also, it is to be noted that, according to further features in preferred embodiments, the construct includes a regulatory elements, such as a promoter, as described herein.
According to still further features in the described preferred embodiments the fruits of the cultivated plants are characterized by an average elongated length-to-width aspect ratio. In certain embodiments, the plants are from the Solanaceae family, including tomato, potato, pepper, eggplant, petunia and the like.
The present invention is based, at least in part, on the discovery that a change in the size and shape of a plant fruit can be achieved by altering the level of SUN expression.
One specific aspect is a transgenic plant cell transformed by a SUN coding nucleic acid expression vector, wherein expression of the nucleic acid sequence in the plant cell results in an alteration in the fruit shape of the resulting plant as compared to a corresponding wild-type variety of the plant cell. In one embodiment, the SUN coding nucleic acid sequence is the SUN from Solannum lycopersicum. 
Another specific aspect is a transgenic plant cell transformed by a SUN antisense coding nucleic acid expression vector, wherein expression of the nucleic acid sequence in the plant cell results in an altered fruit shape of the resulting plant as compared to a corresponding wild-type variety of the plant cell. In one embodiment, the SUN antisense coding nucleic acid sequence is the SUN from Solananum lycopersicum. 
Another specific aspect is an agricultural product produced by any of the transgenic plants, plant parts or seeds described herein.
Another specific aspect is an isolated SUN as described below. In one embodiment, the SUN is SEQ ID NO: 4. Another aspect of the invention is an isolated SUN coding nucleic acid, wherein the SUN coding nucleic acid codes for SUN as described herein.
Another specific aspect is an isolated recombinant antisense expression vector comprising: (a) a promoter, the promoter being functional in a plant cell; and (b) a Solananum lycopersicum SUN antisense coding nucleic acid, the promoter being operably linked to the SUN antisense coding nucleic acid and the antisense coding nucleic acid oriented with respect to the promoter such that the RNA produced is complementary in nucleotide sequence and capable of hybridizing in a stringent manner to mRNA encoding SUN, wherein the SUN antisense coding nucleic acid comprises a nucleotide sequence of at least 15 contiguous nucleotides of SEQ ID NO: 4 compared to a corresponding wild-type variety of the host cell.
Another specific aspect is a method for producing a transgenic plant having altered fruit shape as compared to the corresponding wild-type plant, the method comprising: (a) transforming plant cells by introducing a nucleic acid vector encoding SUN; (b) producing plants from the transformed plant cells.
Another specific aspect is a method for altering the fruit shape of a plant, the method comprising: (a) introducing a nucleic acid vector encoding SUN into a plant cell; (b) regenerating the plant cell into a transgenic plant; and (c) evaluating the change in fruit shape by comparing the plant obtained by introducing the nucleic acid molecule with the size of a corresponding wild-type plant.
According to still further features in the described preferred embodiments, the fruits of the cultivated tomato plant are characterized by an average elongated length-to-width aspect ratio. According to still further features in the described preferred embodiments the fruits of the cultivated tomato plant are characterized by an average fruit shape index greater than 1.
According to still further features in the described preferred embodiments the elongated-fruited tomato is S. Lycopersicum. 
According to still further features in the described preferred embodiments the cultivated tomato is selected from a range of genotypes used in the production of commercial tomato varieties.
According to still further features in the described preferred embodiments there is claimed a tomato fruit derived from the tomato plant. According to still further features in the described preferred embodiments there is claimed a tomato product derived from the tomato fruit.
According to still further features in the described preferred embodiments there is claimed a tomato seed derived from a crossing in which at least one of the parents is the tomato plant.
According to still further features in the described preferred embodiments the tomato seed of claim is a hybrid tomato seed.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Referring now to the FIG. 1A, fine mapping the tomato fruit shape locus sun showed that it was located in a 0.2 cM interval on chromosome 7. Fibre-Fluorescent In Situ Hybridization (Fibre-FISH) experiments using flanking bacterial artificial chromosome (BAC) clones showed that the physical distance in the elongated fruit genotype Sun1642, was approximately 30 kb larger than that of the round-fruited LA15899. However these two BAC clones represented the end points of coverage of the tomato genome as no additional clone from publicly-available genomic large-insert libraries spanned this gap.
To clone the region, bacteriophage lambda genomic libraries were constructed from both the round-fruited LA1589 and the elongate-fruited Sun1642 tomato parents. These libraries were screened initially with probes derived from the ends of the BAC clones used in Fibre-FISH, and subsequently with the ends of recovered phage clones (FIG. 1B).
Comparative sequence analysis of overlapping phage clones showed that the size difference between LA1589 and Sun1642 at the locus was due to the insertion of a 24.3 kb segment present in Sun1642 but absent from LA1589 (FIGS. 1B, 1C).
Genetic analysis indicated the insertion completely co-assorted with the fruit shape phenotype (FIG. 1B). This result strongly implied that the 24.3 kb insertion was the causative mutation that underlied the molecular basis of elongated fruit shape mediated by sun. To determine the origin of the inserted segment, one phage clone end encompassing part of the 24.3 kb segment was used as Restriction Fragment Length Polymorphism (RFLP) probe and hybridized to a set of mapping filters containing genomic DNA from Sun1642×LA1589 F2 progeny. The data showed that LA1589 contained only one copy of this sequence, whereas Sun1642 contained two copies. The copy that was shared between these two tomato accessions mapped to chromosome 10 (see FIG. 5).
Therefore, the data strongly supported the notion that the inserted segment on chromosome 7 originated from chromosome 10. The inventor herein subsequently screened a BAC library constructed from S. lycopersicum cultivar Heinz 170625 and selected one clone from the ancestral location, HBa072D08 for sequence analysis. The data showed that the entire 24.3 kb segment that inserted into chromosome 7 was present on chromosome 10 (FIG. 1C). Interestingly however, despite the near 100% identity at the nucleotide level, the 24.3 kb duplication was not completely colinear with its ancestral copy and comprised another rearrangement (FIG. 1C). The breakpoint of this rearrangement harbored a 3-bp mismatch, which in addition to the rearrangement were the only nucleotide sequence features that distinguished the chromosome 7 and 10 paralogous segments from one another.
Duplication at Sun was the Result of a Retrotransposition Event
The near-identity of the duplication and its ancestor made it likely that the sequence features that resulted in the formation of this copy number variant would be preserved and recognizable. Close inspection of the chromosome 7 and 10 genomic sequences suggested that a Copia-like autonomous retroelement present at both genomic locations might underlie molecular basis of the duplication. The chromosome 10 retroelement element was flanked by identical 398 bp Long Terminal Repeats (LTR) (FIG. 1C, designated in red (dark boxes) as 1 and 2), and a 5 bp target site duplication (TSD) of the signature “GACCT” which are both features of LTR retroelement transposition. Intriguingly, the chromosome 7 region possessed three identical 398 bp LTRs. Two LTRs (LTR 1 and 2) flanked the core retroelement whereas one LTR was further upstream (LTR3) (FIG. 1C).
LTR1 and LTR3 on chromosome 7 flanked the entire duplicated fragment. At the site of the presumed integration and immediately flanking LTR1 and LTR3, a 5 bp motif “ATATT” resembled the TSD of the transposition event (FIG. 1C). In LA1589, which lacked the segmental duplication, only a single copy of the “ATATT” motif was found, which supported the observation that integration of the entire element occurred at this 5 bp motif. Another feature typical of LTR retroelements is the polypurine tract (PPT) which is located immediately upstream of the second LTR (FIG. 1C). The PPT is important for the initiation of the second strand synthesis of the cDNA as well as the subsequent cleavage of PPT from the cDNA. The PPT of the core retroelement was a 15 bp region that started 3 nucleotides upstream of LTR2. A putative PPT that was 70% identical to the PPT upstream of LTR2 was found immediately upstream of LTR3 (PTT* in FIG. 1C). Therefore, the putative PPT was likely to serve as the actual PPT* during transposition of the core element and associated host DNA. This actual PPT* would permit double stranded cDNA synthesis and subsequent integration of the entire retroelement as one single fragment into chromosome 7. Taken together, the sequence features on chromosomes 7 and 10 strongly suggested that the duplication arose via a transposition event mediated by the LTR retroelement.
This novel Copia-like autonomous element was named “Rider” because it traversed the genome and brought along a segment of the host genome on its journey.
Candidate Genes at Sun
The fruit shape phenotype at sun was entirely linked to the inserted segment (FIG. 1B). It was then considered that the elongated fruit shape may be due to genes present on the transposed fragment, or the disruption of a gene at the preexisting locus. Ab initio gene prediction using FGENESH combined with BLAST searches identified four putative genes shared by the chromosome 7 and 10 regions. Present on the duplication were IQD12, SDL1-like, and two hypothetical genes coded HYP1 and HYP2, in addition to the LTR element Rider. In addition, a fifth gene DEFL1 on chromosome 7 was most likely disrupted after the transposition of Rider into this gene (FIG. 1C). IQD12 was most similar to Arabidopsis IQD12, a member of the IQ67 motif-containing plant proteins having calmodulin binding activity26. IQD12 was comprised of 6 exons, all of which were present on the transposed fragment. SDL1-like had high sequence similarity to Nicotiana plumbaginifolia SDL1 gene27 and Arabidopsis ELD128. SDL1-like encoded an 11 exon-containing gene on the parent chromosome 10, but lacked exon 1 and the upstream promoter on the transposed fragment. It now believed by the inventor herein that the transposed copy of the SDL1-like gene was probably not functional and not likely to underlie sun. HYP1, hypothetical 1, was a single exon gene predicted to encode a polypeptide of 350 amino acids having weak similarity to CUC1, an Arabidopsis protein that regulates lateral organ boundary formation29. HYP2 was also a single exon gene, and was predicted to encode a 487 amino acid protein. The best hit to HYP2 was a Solanum tuberosum protein of unknown function (GenBank accession AY737314).
Rider was a single exon retroelement which encoded a 1307 amino acid protein containing the integrase core domain and reverse transcriptase proteins that are required for cDNA synthesis and integration in the host genome. The fifth gene at the locus was DEFL1, consisting of two exons and encoding a secreted defensin protein. The transposition into the intron of this gene strongly suggested that DEFL1 in Sun1642 was inactivated (FIG. 1C). Plant defensins are reported to have anti-microbial and insecticidal properties and are members of a large gene family with up to 317 members in Arabidopsis30, 31. A role in plant development has not been described for any member of this large family.
To test the effect of the sun locus on fruit shape in a homogeneous background and to ascertain whether difference in expression of one of the candidate genes could underlie the phenotype, a set of Near Isogenic Lines (NIL) that differed at sun was generated. The developmental analyses of the NIL in the LA1589 background indicated that ovary shape differences were negligible in floral buds 10 days prior to flower opening (FIGS. 2A and 2B).
At anthesis, ovary shapes began to show significant differences albeit slightly. The most significant differences in shape however were found in developing fruit five days post anthesis (FIGS. 2A and 2B). This result indicated that shape change mediated by sun was manifested primarily following pollination and fertilization, consistent with earlier analyses of the parental genotypes8.
Expression analysis of the five candidate genes during ovary and fruit development revealed much higher transcript levels of IQD12 in the NIL harboring the transposed copy on chromosome 7 in comparison to the NIL lacking the duplication (FIG. 2C). The highest transcript levels of IQD12 were found in young developing fruit five days after anthesis. However, transcript levels of the disrupted gene DEFL1 also significantly differed in the NIL and showed an expression pattern that was essentially the inverse of IQD12: when IQD12 was expressed, DEFL1 was not and vice versa (FIG. 2C).
Reverse transcription-PCR analyses failed to detect DEFL1 transcript in the NIL that carried the duplication. This finding strongly indicated that DEFL1 function was abolished as a result of Rider's transposition into this gene. The transcript levels of the other genes transposed by Rider, SDL1-like, HYP1, and HYP2 were not altered or undetectable and were deemed less-likely candidates of the SUN gene.
The fruit shape phenotype controlled by sun was dosage-dependent, e.g. NIL plants that were heterozygous at the sun locus exhibited a fruit shape phenotype between that of both parents which was indicative of a gain-of-function mutation (FIG. 2D). To investigate whether transcript levels of IQD12 and DEFL1 were also affected by a dosage effect, total RNA was isolated from developing fruits of individual plants homozygous for either the Sun1642 or LA1589 allele, or heterozygous. Northern blot analyses showed that IQD12 was indeed expressed approximately two-fold higher in individuals homozygous for the transposed fragment than it was in heterozygous plants. Similarly, DEFL1 was expressed about two-fold higher in homozygous individuals lacking the transposed fragment than in heterozygotes (FIG. 2D).
It was also observed that there was a similar dosage effect on fruit shape and expression levels of IQD12 and DEFL1 in the NIL in the Sun1642 background (FIG. 7).
Thus, only one gene was expressed in either homozygous situation, indicating that expression of IQD12 and DEFL1 were mutually exclusive and dosage-dependent. Moreover, these results suggested that the transposition event placed IQD12 in a genome environment in which it was now under the cis-regulatory control of factors that normally conferred high levels of DEFL1 expression in developing fruit.
Complementation of the Fruit Shape Phenotype
To determine whether genomic fragments encompassing IQD12 were capable of imparting an elongated phenotype to fruits, the entire insert from two overlapping Sun1642λ genomic clones were subcloned into an Agrobacterium tumefaciens binary vector and transformed into LA1589 and the round-fruited NIL in the Sun1642 background. These two clones, pHX2 and pHX4, harbored the full length IQD12 gene, including the promoter that was shared with the ancestral gene and the LTR, but contained different 5′ as well as 3′ end points (FIG. 3A).
In the LA1589 background, most of the primary plants transformed with the pHX4 construct (T1) expressed IQD12 at very high levels, whereas this gene was very low or not at all expressed in the pHX2 primary transformants (FIG. 3B and FIG. 10—Table 1).
Moreover, pHX4 transformed T1 plants exhibited a significantly greater fruit shape index, whereas those transformed with the pHX2 construct did not. Regression analysis confirmed that there was a highly significant correlation between IQD12 transcript levels and fruit shape index which was in turn correlated to transformation with the pHX4 construct (FIG. 3C). The transformation of the same constructs into the round-fruited NIL in the Sun1642 background produced similar results: lines transformed with pHX4 displayed a larger fruit shape index in comparison to control lines whereas none of the lines carrying pHX2 displayed significantly elongated fruit (FIG. 11—Table 2).
Increased expression of IQD12 and increased fruit shape index were inherited to the T2 generation, and cosegregated with the presence of the pHX4 construct (FIG. 3D and FIG. 8A). The transcript levels of IQD12 and fruit shape index of the T2 plants homozygous was compared to the pHX4 transgene to that of the NIL. These results indicated that for most transgenic plant families, the fruit shape index and the IQD12 transcript levels were nearly restored to the levels displayed by the NIL carrying the transposed segment (FIG. 3D, FIG. 10, FIG. 13).
Thus, the data obtained from the plants transformed with pHX4 indicated that the control of fruit shape in tomato was regulated at the level of transcription of IQD12. In addition, an important cis-element located in the 3.2 kb region upstream of DEFL1 was sufficient to drive high levels of IQD12 expression in the developing fruit as this region was present in the pHX4 transformants expressing IQD12 and absent from the pHX2 transformants that did not express this gene to appreciable levels (FIGS. 3A-3C).
Also, the results from the pHX2 and pHX4 transformations demonstrated that the SDL1-like and HYP1 genes were unlikely candidates in affecting fruit shape because these genes were present on the pHX2 construct that had no affect on fruit shape (FIGS. 3B, 3C).
Although transcript levels and fruit shape index were significantly increased in the transgenic lines carrying pHX4, they were not entirely restored to the levels of that in the NIL carrying the transposed segment. Thus, it was possible that additional cis-elements in the DEFL1 5′ upstream region beyond the 5′ end point of this construct were necessary to fully recover the fruit shape phenotype or that other genes were required (FIG. 3D and FIG. 8).
To determine whether IQD12 alone was sufficient to confer an elongated shape to tomato fruit, this gene was overexpressed in the round-fruited LA1589, and used an RNA interference (RNAi) strategy to knock-down expression in LA1589 carrying the transposed segment and exhibiting elongated fruit. Six of 13 LA1589 lines transformed with IQD12 under control of the constitutive CaMV 35S RNA promoter bore extremely elongated fruit and expressed IQD12 to high levels (FIG. 3E; FIG. 12—Table 3, FIG. 9A).
Since fruit and seed set were significantly reduced in the transgenic lines that overexpressed IQD12, these lines were maintained by using the transgenic pollen to pollinate wild type LA1589 pistils. The resulting T2 plants showed that the extremely elongated fruit shape and associated high levels of expression of IQD12 were inherited in the next generation in the presence of the transgene (FIG. 3B, FIG. 13—Table 3).
Complimentary experiments using an RNAi strategy in which an IQD12 inverted repeat construct was transformed into NILs that carry the transposed segment resulted in reduced fruit shape index and decreased IQD12 expression in seven of the 14 primary T1 transformants (FIG. 3F, FIG. 13—Table 4, FIG. 9C).
For three of these transformants, the fruit shape phenotype was essentially indistinguishable from the round-fruited non-transformed NIL (FIG. 12—Table 3).
Progeny testing of the seven primary transgenic plants that bore rounder fruit and comparison to the non-transformed NIL confirmed that reduction in IQD12 transcript levels resulted in rounder fruit which was dependent on the presence of the transgene (FIG. 12—Table 3, FIGS. 9D, 9E).
The results show that greatly enhanced transcript levels of IQD12 can control fruit shape phenotype controlled by the sun locus. As IQD12 conferred an elongated phenotype to tomato fruit controlled by the sun locus (renamed IQD12 gene as SUN).
SUN is a Member of the IQD Family of Proteins Found in Plants
The most prominent feature of the IQD family is a domain of 67 amino acids, the IQ67 domain, which is shared by all members and to date has only been found in plants26. The IQ67 domain of the founding member of this family, AtIQD1 is shown to bind calmodulin and localized in the nucleus32. Overexpression of AtIQD1 leads to increased glucosinolate levels in Arabidopsis32. Based on the shared domain structure and phylogenetic analysis, SUN was most closely related to AtIQD12 (FIG. 4) which was part of a subgroup of IQ67 domain-containing proteins that clustered in the subfamily II clade26. However, using the criterion of conservation of intron position, SUN was most closely related to AtIQD11 which is a member of the same subfamily II (FIG. 4A). Like SUN, AtIQD11 harbored six exons, including the first exon which did not code for part of the protein. However as noted by Abel et al26, the Arabidopsis IQD12 displays an unusual genome structure because this gene is the only IQ67 domain member that lacks a highly conserved intron positioned within the IQ67 domain. Thus, it is possible that intron removal from the Arabidopsis IQD12 gene occurred subsequent to the divergence of this family and that SUN and AtIQD12 could share a common ancestor. Because of its closest similarity at the amino acid level, SUN was considered the likely tomato ortholog of the Arabidopsis IQD12. Plant EST and genomic database searches identified other IQ67 domain-containing proteins that were closely related SUN. None of these however, correspond to members of the monocotyledon lineage of plants including rice. The phylogenetic tree constructed using the IQ67 domain of these proteins indicated two clusters; one containing the Arabidopsis IQD11 and the other IQD12 (FIG. 4B). One tomato member in the IQD11 Glade and two tomato members in the IQD12 clade were identified. In addition, a close homolog of SUN was found in potato (BQ116231).
Presence of Sun in Other Tomato Varieties
Next, it was determined whether the sun locus played a role in the fruit shape phenotype that is observed in older accessions (heirloom) as well as other more recent tomato introductions. Although the origin of open pollinated heirloom varieties was often unclear, a few were likely to originate from Europe. The heirloom Spitze was likely brought over from Romania, whereas Howard German was likely from the “old country” and was presumably of German origin. Roma VF was introduced in 1963 as the Verticillium and Fusarium-resistant version of the heirloom Roma that originated from Italy. Banana Legs, Long John, Jersey Devil and Yellow Stuffer were more recent introductions although they were often listed with the heirloom and open pollinated varieties. Sun1642, M82, TA496 and Heinz1706 represented experimental processing tomato varieties. These genotypes were used for the construction of the tomato introgression lines (M82)33, the large EST collection (TA496)34, or were the resource for the tomato genome sequencing project (Heinz1706; SGN)25.
To determine whether these accessions carried sun, all the varieties were crossed to S. pimpinellifolium LA1589. Via marker-assisted selection, plants that were homozygous S. lycopersicum, heterozygous and homozygous LA1589 at sun were identified from each of the resulting F2 populations. The shape of mature fruit was analyzed in these plants, and the correlation of shape and marker score were assessed (FIG. 5A).
The results showed that varieties in which fruit shape segregated at sun (denoted “+”) exhibited larger fruit shape indices compared to varieties that didn't segregate for fruit shape at sun (denoted “−”) (FIG. 5A). Moreover, Southern blot hybridization using a probe derived from the duplication showed that varieties segregating for fruit shape at sun (FIG. 5A) carried the transposed copy of SUN (FIG. 5B).
Expression analysis confirmed that the genotypes harboring the duplication expressed SUN at high levels whereas those genotypes lacking the duplicated gene did not express SUN (FIG. 5C).
Genotyping these varieties for ovate which was another locus that controlled elongated fruit shape showed that Long John, Roma VF, and Heinz1706 carried the allele that conferred an elongated pear shape to fruit. Fruit shape in the less elongated and round varieties, M82 and Yellow Stuffer, respectively, did neither carry the elongated allele of sun nor ovate. Thus, based on the fruit shape index of the mature fruit, the varieties exhibiting the more elongated fruit carried sun, whereas the varieties displaying a less elongated shaped fruit carried ovate. Accessions that bore round or slightly oval shaped fruit, M82 and Yellow Stuffer, carried neither locus whereas Long John that carried the most extremely elongated tomato fruit had both the elongate shaped sun and ovate alleles (FIG. 5A). These results indicated that the transposed segment that gave rise to the sun locus was present in a subset of varieties carrying elongated fruit, some of which were heirloom and others that were modern types. Therefore, once the sun locus arose, it did spread through the germplasm and was maintained until present time.
Discussion
Provided herein is evidence of an unusual mutation event that resulted in an elongated fruit phenotype found in heirloom and modern varieties of tomato. The mutation was the result of a duplication of SUN into another genomic context. The new location of SUN on tomato chromosome 7 permitted high expression of this gene in developing ovaries and fruits which resulted in an elongated fruit shape. In plants, a translocation resulting in a copy number variant that resulted in phenotypic change have not been described to date.
SUN on chromosome 7 is positioned downstream of the promoter of DEFL1. Transformation with genomic constructs that harbor different promoter lengths indeed implied that the DEFL1 promoter acted as an enhancer for transcription of SUN.
Although LTR are known to carry promoter and enhancer activities, it is unlikely that Rider's LTR acted as an enhancer of SUN transcription since this LTR was present on both genomic constructs that were transformed into plants, and only one of them resulted in complementation of the fruit shape phenotype.
The gain-of-function mutation resulted from a transposition event mediated by the autonomous LTR-retroelement Rider. The transposition of the core retroelement was associated with 3′ transduction of nearby genes as well as a second rearrangement that moved SUN from a few kb upstream of Rider to 20 kb downstream of this autonomous element. To explain how the transposition, associated gene transduction and second rearrangement occurred, a model is described in FIG. 6A.
Transcription of Rider on chromosome 10 began, as in all LTR retroelements, in LTR1. Typically, transcription would stop in LTR2; instead transcription read through occurred past the second LTR and continued far beyond into the flanking genomic region (FIG. 6B). In the first intron of the SDL1-like gene, the RNA polymerase switched to a region 3′ of IQD12. Presumably, such a template switch would have required that these two regions were in proximity of one another. The 5-bp direct repeat “GCAGA” on each side of the breakpoint suggested that this was where the template switch occurred. Thereafter, elongation of the transcript continued until termination in LTR1 (FIG. 6B), resulting in a large mRNA molecule flanked by part of two LTRs, the R segment and U5 region at the 5′ end of the mRNA and the R segment and U3 region at the 3′ end of the mRNA37 (FIG. 6C). Reverse transcription of the large mRNA, followed by second strand synthesis initiated at PPT*, resulted in a 24.3 kb element harboring three identical LTR; LTR1 and LTR3 flanking the entire element and LTR2 as an internal or solo LTR (FIG. 6D). This giant element then inserted into chromosome 7 (FIG. 6D) resulting in the genome structure that is found at the sun locus (FIG. 6E).
Several lines of evidences are in agreement with the proposed transposition mechanism. First, the 24.3 kb duplicated fragment is encompassed by two LTRs of Rider, indicating that Rider was directly involved in the duplication (FIG. 6C). Second, two signature TSD sequences “ATATT” are flanking the two outer LTRs (LTR1 and LTR3), strongly suggesting the entire fragment moved by a single transposition event (FIGS. 6C-6E). This explanation of the transposition is also supported by the observation that the original TSD of the retroelement on chromosome 10, “GACCT”, was copied and now borders LTR2 and LTR3 on chromosome 7 (FIG. 6E). Also, this model explains the retention of the introns of IQD12 and SDL1-like genes since they are in the antisense orientation with regard to Rider. Therefore, the introns were not recognized and removed by the splicing machinery after RNA synthesis. Rider's 3′ gene transduction and subsequent transposition event is very unusual since parts of the two flanking LTRs are absolutely required for reverse transcription and double stranded DNA synthesis of LTR retrotransposons37. Therefore, this element could not have been competitive for transposition, unless parts of the LTRs flanked the RNA molecule. Thus, transcription of a LTR retroelement past LTR2 will only result in an effective transposon if the transduction terminates in another LTR, as occurred with Rider via the template switch. The transcription read through and 3′ transduction, followed by successful transposition is more commonly observed in non-LTR retroelements for which transcription does not need to end at a particular site.
Examples of 3′ transduction and transposition to a second site are found certain L1-mediated retrotransposition events38-40. In fact, in humans 1% of the genomic sequence is estimated to have been transduced by such a mechanism that was mediated by the non-LTR L1 element21. An interesting example of a 3′ transduction event which is perhaps most analogous to the transposition of Rider is mediated by the non-LTR SVA retrotransposon41. Duplications of human AMAC1, encoding acyl-malonyl condensing enzyme 1, were mediated by this element via a 3′ sequence transduction mechanism and the entire gene including the promoter, which was located downstream of the SVA element, was duplicated and inserted into three different chromosomal regions along with the SVA retroelement41. Moreover, two of the three AMAC1 gene duplicates are differentially expressed in different tissues relative to the original gene, and thus new functionality may have been created for each of the AMAC1 duplicates following its retrotransposition41. However, the gene transduction and transposition event mediated by Rider is extremely rare for LTR retroelements given that those elements require flanking LTR for proper transposition.
As disclosed herein, the cloned SUN is one of the major genes controlling elongated shape in tomato. SUN encodes a IQ67 domain-containing protein which are found in diverse plant species from gymnosperm to angiosperm plants and even in moss26, 32. Moreover, SUN controls fruit shape in other accessions and therefore presents an important mutation in the tomato germplasm. The founding member of this family of proteins is the Arabidopsis IQD1 protein. IQD1 plays a role in glucosinolate production, binds calmodulin, and is nuclear localized32. The function of the other Arabidopsis members remains unknown. Glucosinolates are not produced by Solanaceous species and therefore, SUN is unlikely to affect the production of those metabolites in tomato. However, AtIQD1 is also thought to play a role in the transcriptional regulation of several cytochrome P450 genes including CYP79B3 and CYP79B232, 42. CYP79B3 together with CYP79B2 catalyzes the conversion of tryptophan into indole-3-acetaldoxime (IAOx) in tryptophan-dependent auxin biosynthesis in Arabidopsis43-45. Although it is not clear whether AtIQD1 plays a role in the homeostatic control of auxin biosynthesis, the over and under expressing of AtIQD1 disturb plant growth slightly32. Regulating the production of and sensitivities to secondary metabolites as well as plant hormones is a common strategy that plants use to coordinate plant growth and responses to environmental and developmental pressures. Since the IQD protein family widely exists in the plant lineage, these proteins may play important roles in the life cycle and may affect diversity in plant form in other species. Their diverse amino acid sequences and protein structures may imply extensive selection pressures that persisted during their evolution26. Therefore, the IQD genes may have evolved into new functions to respond to selection pressures specific to each plant species.
In tomato, overexpression of SUN resulted in extremely elongated and often seedless fruit. These features are reminiscent of parthenocarpic and elongated and pointed fruit that are resulting from the expression of controlling auxin production in ovules.
The extremely elongated fruit shape and the lack of proper seed development when SUN is overexpressed, in addition to its potential biochemical function suggest that this protein may affect auxin levels or distribution in the fruit (FIG. 3E). Consequently, it is possible the involvement of SUN in shape variation is through the regulation of auxin homeostasis, thereby affecting the patterning of the fruit.
Examples
Methods
Plant Materials
The high-resolution screen to identify recombinants delineating the fruit shape locus was described previously9. Near Isogenic Lines (NILs) differing at sun were constructed in both Sun1642 and LA1589 backgrounds by sequential backcrosses to the recurrent parent using marker-assisted selection.
Primary transformants (T1) carrying various constructs were generated through Agrobacterium-mediated transformation at Ralph M. Parsons Foundation Plant Transformation Facility, College of Agricultural and Environmental Sciences, University of California (Davis, USA).
Selection of T2 transgenics was done by Southern blot analysis and homozygosity was confirmed by genotyping the progenies using KanR specific primers EP551 and EP552 (pHX4 transgenic lines 06S496-501 and P35S:IQD12 transgenic lines 07S15-18) or using primers EP687 and EP688, which amplify the chalcone synthase intron in the RNAi vector, pFGC5941.
Primer sequences are listed in FIG. 13—Table 4 showing SEQ ID NOS:14-41. Plants used in this example were grown in the greenhouse at 25-32° C., 40-60% humidity, and supplemented with 200 μM m−1s−1 of light from high pressure sodium lamps. The RNA collected from the plants shown in FIG. 5 were field-grown during the summer of 2005 in Wooster, Ohio, USA.
Microscopy
Ovaries were harvested at 10 days pre anthesis, fixed and embedded in LR white resin, sectioned (5 μm) and stained with toluidine blue. Developing ovaries were visualized on a Leica DM IRB light microscope (Leica Microsystems Heidelberg GmbH, Germany) coupled to a digital camera (Optronics 60806, Olympus America Inc. USA).
Fresh anthesis stage ovaries and 5 dpa fruits were sectioned longitudinally and photographed using a Leica MZFLIII dissecting microscope equipped with an attached digital microscope camera (SPOT RT KE, Diagnostic Instruments, Inc. USA).
Construction and Screening of Phage Libraries.
Bacteriophage λ genomic libraries were constructed in the vector λ Fix® II using the components of the λ FixII Gigapack III XL kits from Stratagene (La Jolla, Calif.). Partially Sau3AI-digested genomic DNA of Sun1642 and LA1589 was ligated into XhoI-digested lambda FIX II vector. Complete details of library construction are available on the Stockinger Lab Web site (http://www.oardc.ohio-state.edu/stockingerlab/). The first probe used to screen the phage libraries and to initiate the chromosome walk was generated from BAC end Lp81B9-F using a genome walking approach and components of the GenomeWalker kit (Clontech, California, USA). For information on these BACs and end sequences, see Van der Knaap et al.,9. The fragment amplified with primers EP8 and EP9 mapped approximately 2 kb away from BAC end Lp81B9-F (FIG. 13—Table 4 for primer sequences).
Sun1642 phage clone EK36 was recovered after screening both parental libraries. At the other end of the gap, BAC end Lp61O2-F9 was present in multiple copies in the genome and could therefore not be used in genomic library screens. Instead, Le33O1-R9, which was uniquely represented in the genome, was used for the phage library screens resulting in several clones that were identified from both libraries. These screens were followed by sequential screening with 32P-labeled probes from unique end sequences of overlapping phage clones until the region was covered.
Sequencing of Large Insert Clones and Construction of the Sun Locus Contig.
Minimal overlapping λ clones were identified and the entire inserts were subcloned into the NotI site of pGEM11fz(+) vector. Shotgun libraries were generated from the plasmid inserts and sequenced using M13 forward and M13 reverse primers; complete details of all procedures are available on the Stockinger Lab Web site (http://www.oardc.ohio-state.edu/stockingerlab/). BAC clone HBa072D08 corresponding to the ancestral locus was identified from Heinz1706 library25 using the phage clone end of EK36 that maps on the duplication (primers EP45 and EP46, FIG. 13—Table 4).
Sequencing was conducted at the Genome Sequencing Center, Washington University in St. Louis, Mo., USA, with the exception of clone, EK58, which was sequenced at the Purdue Genomics Core Facility, Purdue University. BAC HBa072D08 clone was shotgun subcloned, sequenced, and finished to completion at the Genome Sequencing Center, Washington University in St. Louis. The finishing of all phage clone insert sequences was done by primer walking using the large clones or the subclones as sequencing template at the Molecular and Cellular Imaging Center at Ohio State University, Wooster, Ohio on the ABI Prism 3100 Applied Biosystem, Foster City, USA. Sequences were assembled into one contig per clone using Sequencher 4.1.4 (Gene Code Corporation, USA) and manually corrected and edited. Questionable base calls in the assembled sequences were checked by PCR amplification using a genomic DNA template and sequencing of the PCR product.
Sequences of overlapping clones were assembled into a single contig representative for the two sun alleles present in Sun1642 and LA1589. The one exception was an approximate 3 kb gap in the Sun1642 contig (from nucleotides 27,755 to 30,601 in the deposited EF094940 GenBank sequence). This region was not recovered from the phage libraries because of 100% identity to the ancestral chromosome 10 locus. In this instance the sequence of the gap was inferred from the chromosome 10 HBa072D08 BAC sequence harboring the ancestral version. To confirm that these nucleotide sequences were present in the transposed chromosome 7 duplication, Southern blot hybridizations were performed using a PCR amplified fragment (primers EP293 and EP294) across this region as probe (FIG. 13—Table 4). This segment maps in the gap and showed two bands of the expected sizes on a Southern blot that carried Sun1642 DNA and digested with ScaI and XbaI (data not shown). Overlapping phage clones used in Fiber FISH experiments also showed that the distances between the clones were as predicted based on the complete sequence of the chromosome 10 locus (data not shown).
Sequence Annotation
Following assembly, the sequences were analyzed using the ab initio gene prediction program FGENESH. Final annotation of the genomic sequences using NCBI and TIGR databases was done at TIGR in the group of Dr. Robin Buell. Repetitive elements in the sequenced regions were identified by their repetitive features and by the presence of transposase coding sequences. Specifically, sequences were used as queries in Blast searches against tomato genomic sequences using a 1 kb window. The tomato genomic sequences were downloaded from SOL Genomics Network (27 Mb total size). Genomic fragments with five or more hits in the search were manually examined for features associated with transposable elements, including terminal sequence, terminal inverted repeats, direct repeats and target site duplications. All genomic sequences were searched against NCBI non-redundant protein database (Feb. 15, 2007) for proteins related to autonomous transposons. Rider was the only autonomous element detected in this region.
Constructs for Plant Transformation
Transformation constructs pHX2 and pHX4 were made by subcloning the entire phage subclone insert of plasmids pEK59 and pEK60, respectively (released by NotI and blunted-ended using Klenow) into the Klenow-blunted ended BamHI-digested binary vector pCIB10G46. End points of the clones are indicated in FIG. 3A.
The RNAi:IQD12 construct, pHX8, was generated by cloning 512 bp fragments of the IQD12 cDNA (from nucleotide 16,154 to 16,646 of the genomic sequence EF094940) that was amplified using primer EP527 and EP528 from reverse transcribed mRNA, in the sense and antisense directions into pFGC594147.
To over expresses IQD12, a 1.4 kb fragment of IQD12 cDNA (corresponding to nucleotide 13,460-16,280 of the genomic sequence EF094940) was amplified from reverse transcribed mRNA using primer EP519 and EP520 and subcloned between the CaMV 35S RNA promoter and NOS terminator of pCIB71046 generating pEK67. Promoter, insert and terminator were released from the vector by KpnI and XbaI digestion and were subcloned into the corresponding sites of binary vector pCIB10G creating pEK69. Plasmid constructs were introduced into Agrobacterium tumefaciens strain LBA4404 using electroporation. Plasmid pHX8 was transformed into the NIL carrying the Sun1642 allele in the LA1589 background. Plasmid constructs pHX2, pHX4, and pEK69 were used to transform round-fruited NIL plants in both the LA1589 and Sun1642 backgrounds.
Northern and Southern Blot Analysis
Tissues for RNA extraction were harvested and immediately frozen in liquid nitrogen. Flower buds at 10 days preanthesis were identified as follows. Using a dissection microscope, the floral bud for which the sepals had just enclosed the remaining floral meristem was located on the inflorescence. Counting 4 to 5 buds upwards (older buds) on the same inflorescence marked the style and ovule initiation stage which occurred approximately 10 days prior to flower opening. To collect fruit five days post anthesis, recently opened flowers (within 24 hours) were hand pollinated to ensure sufficient seed and fruit set and the developing fruit was harvested 5 days later. Total RNA was isolated using Trizol® reagent (Invitrogen Inc. USA) following the recommendations of the manufacturer. Ten μg of total RNA from each sample was size-fractionated on a 1.2% agarose-formaldehyde gel prior to transfer to Hybond N membrane filters. A detailed protocol of the Northern and Southern blot hybridization are found at http://www.oardc.ohio-state.edu/stockingerlab/. Gene-specific probes were generated using a 3 cycle PCR step in the presence of [α-32P]dCTP as described previously9.
After hybridization, filters were washed at high stringency 0.1×SSC, 0.1% SDS at 65° C. for 20 minutes. The blots were exposed to phosphorimager screens and visualized using the Storm 840 scanner (GE Life Sciences, USA). For quantification of gene expression, signals were quantified by ImageQuant 5.0 (Molecular Dynamic System Inc. USA) and normalized to the expression of the tomato eIF4α6 gene. Probe sizes and primers used for amplification of probe templates are listed in FIG. 13—Table 4.
Amino Acid Alignment and Phylogeny of SUN
Genes that are closely related to tomato IQD12/SUN were identified by BLAST searches using TBLASTN function against the public databases including the Solanaceae Genomics Network (SGN), Joint Genome Institute; and NCBI. The Arabidopsis thaliana IQ67-motif containing genes were directly retrieved from NCBI26. ClustalX (v1.83) was used for sequence alignments. The unrooted phylogenetic tree was also generated using ClustalX; bootstrap values represent 1000 trials. MEME v3.5.4 was used to predict motifs in IQD12/SUN and the 33 IQ67 Arabidopsis thaliana homologs26. Only motifs with p-value smaller than 1.0e-5 are shown.
Expression of SUN in Different Tissues:
FIG. 14A shows the Gene structure at the sun locus. The 24.7 kb duplication, including SUN, disrupted the expression of DEFL1. FIG. 14B shows the expression of SUN and DEFL1 in floral tissues. Northern blot containing RNA isolated from floral organs at the time of anthesis. The blot was sequentially probed with SUN, DEFL1 and lastly with eIF4α-6 as loading control. Se, sepal; Pe, petal; St, stamen; Ov, ovary. Tissues were isolated from the LA1589 and Sun1642 NILs that differ at the sun locus. ee, containing an extra copy of SUN; pp, lacking the extra copy of SUN.
FIG. 14C shows the expression of SUN and DEFL1 in different tissues. Tissues were isolated from the LA1589 and Sun1642 NILs that differ at the sun locus. ee, containing an extra copy of SUN; pp, lacking the extra copy of SUN. R, root; H, hypocotyl; C, cotelydon; L, leaf; S, shoot apex. In lines carrying the duplication, SUN is highly expressed in sepals, ovaries, hypocotyl and shoot apex. The ancestral SUN gene is expressed in roots and at low levels in other tissues. DEFL1 is expressed in the same tissues as the duplicated copy of SUN in plants that lack the 24.7 kb duplication (pp, lacking an extra copy of SUN). This indicates that the promoter of DEFL1 is driving expression of the duplicated copy of SUN.
Fruit Shape Index and SUN Expression Changes During Tomato Flower and Fruit Development:
FIG. 15A shows the fruit shape index (length/width ratio) as a function of the number of days post anthesis. Fruit shape changes overlay the fruit/seed developmental landmarks indicated above the graph. The black triangles represent the fruit shape indices of the near isogenic lines (NILs) carrying two copies of the SUN gene whereas the grey circles represent the NILs with only one copy of SUN. The largest difference in fruit shape index is achieved at fruit landmark 3 and 4, coinciding with the landmarks 4-16 cell and globular stage of the embryo. Fruit shape index were collected from three inflorescences per plants of five for each genotype. Data shown are mean±standard error.
FIG. 15B shows the SUN expression in the developing fruits of LA1589 NILs. Northern blots were performed on LA1589ee (carrying two copies of the SUN gene) and LA1589pp (carrying one copy of SUN). Total RNA was extracted from pooled tissues of five plants per genotype and hybridizations were conducted with SUN as probe. SUN expression is very high starting at anthesis until 20 days post anthesis. Its expression has dropped dramatically 25 days post anthesis which is prior to the fruit ripening and seed germination stage.
FIG. 15C shows the SUN expression in floral buds of LA1589 NILs. Northern blots were performed on total RNA isolated from entire flowers or buds at the times indicated above the lanes (in days). The “0” timepoint denotes anthesis, the other values indicate days prior (−) or post (+) anthesis. SUN's expression is low 2 days pre-anthesis but increases dramatically until 2 days post pollination in the lines carrying two copies of SUN (LA1589ee). The increase in SUN expression precedes the change in fruit shape index shown in FIG. 15A. In the lines carrying only one copy of SUN (LA1589pp), SUN expression is low, however DEFL1 expression in LA1589pp follows a similar kinetic as SUN expression in the LA1589ee indicating that the DEFL1 promoter drives SUN expression (see also FIG. 14).
The Effect of SUN on Leaflet Shape:
FIG. 16A shows the leaflets of cultivated tomato. FIG. 16B shows leaflets of the wild relative S. pimpinellifolium accession LA1589. The leaves shown are from plants without the extra copy of SUN (pp), with the extra copy of SUN (ee) or SUN expressed under the constitutive 35S promoter (ox). The most notable feature is the pointed shape of the leaf and increased serrated margins when SUN is expressed (compare pp and ee). These features are accentuated when SUN is overexpressed (compare ee and ox). These results indicate that in addition to fruit shape, leaf shape is dramatically altered as well.
Effect of Overexpression of SUN on Ovary, Fruit and Compound Leaf Shape in Tomato:
FIGS. 17A-17C show the S. pimpinellifolium LA1589 background. Ovary shape of anthesis-stage flowers (FIG. 17A), fruit shape (FIG. 17B) and compound leaf shape (FIG. 17C) of wild type (LA1589pp) and 5 independent transformants. Note the very elongated slender shape of the ovary, fruit and the twisted shape of the leaves.
FIGS. 17D-17F show the S. lycopersicum Sun1642 background. Ovary shape of anthesis-stage flowers (FIG. 17D), fruit shape (FIG. 17E) and compound leaf shape (FIG. 17F) of wild type (Sun1642pp) and several independent transformants. When SUN is overexpressed under control of the 35S promoter, the fruit shape is already determined at the time of anthesis. Moreover, leaflets and compound leaf shape is also greatly affected when SUN is overexpressed. The leaves are twisted and the leaflets are more pointed in shape.
Cell Size and Number Differences in the Longitudinal Direction of LA1589 NIL Fruit Differing at Sun:
FIG. 18A shows the length of different parts of the fruit at 5 days post anthesis. All fruit parts are more elongated in the presence of SUN. FIG. 18B shows that cell size is only significantly different in the distal end of the fruit. FIG. 18C shows that the ratio of fruit length and cell size shows that the septum and proximal end of the fruit have significantly more cells. Thus the effect of SUN is on increased cell division predominantly in the septum of the fruit thereby increasing fruit length.
Cell Size and Number Differences in the Latitudinal Direction of LA1589 NILs that Differ at Sun:
FIG. 19A shows the width of the fruit at 5 days post pollination. Total fruit and septum width are significantly smaller in the NILs carrying the SUN duplication. FIG. 19B shows that the eell number in the septum is significantly lower in the NILs carrying the SUN duplication. FIG. 19A-FIG. 19C shows that the cell size is not significantly different in the septum or pericarp. The results shown in FIGS. 18A-18C and FIGS. 19A-19C demonstrate that SUN controls directional cell division predominantly in the septum and proximal end of the fruit. High expression of SUN leads to increased cell division in the longitudinal direction and reduced cell division in the latitudinal direction. This suggests that SUN can affect the shape of any organ in any plant species depending on where and when the gene is expressed.
FIG. 20 shows the stem structure of S. lycopersicum cv Sun1642pp (without the SUN duplication and carrying round fruit) and Sun1642pp overexpressing SUN.
A cross section of the tomato stem at the sixth leaf of round-fruited tomato (pp, control) and overexpressors (35S::sun). The sections were hand cut and stained with Toluidine Blue. The xylem tissues of the vascular bundles stain blue. Note the round stem shape exhibited by the overexpressing lines compared to the triangular stem shape in the control plants. Also note the expansion of the xylem in the overexpressing lines at the expense of the pith cells (in the center of the stem). Thus, in the cultivated background, overexpression of SUN leads to altered stem structure and changes in cell identity where pith cells become xylem cells.
FIG. 21—Table 5 shows the near isogenic lines that differ at sun in both the Sun1642 and the LA1589 background show changes in fruit shape index, leaflet shape index (see also FIG. 16), sepal and ovary shape index and to a lesser extent petal shape index, and seed weight. Fruit weight, number of seed per fruit, hypocotyl and internode length is not altered. The fact that SUN does not affect fruit weight but only the shape strongly indicates that the gene acts to redirect growth without increasing growth. Again, this finding shows that SUN may be able to alter direction of growth of any plant organ.
FIG. 22—Table 6 shows that leaf shape, fruit shape, seed number per fruit, seed and fruit weight are similar in the line expressing SUN under its own promoter compared to the NIL carrying the SUN gene duplication. This shows that only SUN but neither DEFL1 nor one of the HYP genes (hypothetical, see FIG. 14) affect shape of plant organs and seed weight.
Expression of SUN in Additional Varieties of Tomatoes and Other Plants
The inventor has also established which of over 300 tomato varieties carry the SUN gene and has studied its expression pattern throughout fruit development.
SUN Expression in Arabidopsis 
The inventor has also expressed the tomato gene in Arabidopsis. 
SUN Expression in Other Plants
SUN can be expressed in other plants in addition to Arabidopsis using a constitutive promoter that works in all plants.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
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## Claims

1. A transgenic Lycopersicon plant transformed with an isolated nucleic acid sequence which encodes SEQ ID NO: 4 and an isolated DEFL1 promoter operably linked to the isolated nucleic acid sequence which encodes SEQ ID NO: 4.

2. The transgenic Lycopersicon plant of claim 1, wherein the transgenic Lycopersicon plant is a near isogenic line.

3. A plant part, plant seed, plant cell, or the transgenic progeny of the transgenic Lycopersicon plant of claim 1.

4. The plant part, plant seed, plant cell, or the transgenic progeny of claim 3, wherein the transgenic Lycopersicon plant is selected from the group consisting of: Lycopersicon esculemm and Lycopersicon pimpinelliforlium.

5. A method for making a transgenic Lycopersicon plant having altered fruit shape, comprising breeding the plant of claim 1 with another Lycopersicon plant.